U.S. patent application number 10/659800 was filed with the patent office on 2004-04-22 for diacylglycerol o-acyltransferase.
Invention is credited to Cases, Sylvaine, Erickson, Sandra K., Farese, Robert V. JR., Smith, Steven.
Application Number | 20040078836 10/659800 |
Document ID | / |
Family ID | 27488655 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040078836 |
Kind Code |
A1 |
Farese, Robert V. JR. ; et
al. |
April 22, 2004 |
Diacylglycerol O-Acyltransferase
Abstract
Nucleic acid compositions encoding polypeptide products with
diglyceride acyltransferase activity, as well as the polypeptide
products encoded thereby and methods for producing the same, are
provided. The subject polypeptide and nucleic acid compositions
find use in a variety of applications, including research,
diagnostic, and therapeutic agent screening applications, as well
as in treatment therapies and in the production of
triacylglycerols.
Inventors: |
Farese, Robert V. JR.; (San
Francisco, CA) ; Cases, Sylvaine; (Belmont, CA)
; Smith, Steven; (San Francisco, CA) ; Erickson,
Sandra K.; (San Francisco, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
27488655 |
Appl. No.: |
10/659800 |
Filed: |
September 10, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10659800 |
Sep 10, 2003 |
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10040315 |
Oct 29, 2001 |
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10040315 |
Oct 29, 2001 |
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09339472 |
Jun 23, 1999 |
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10040315 |
Oct 29, 2001 |
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PCT/US98/17883 |
Aug 28, 1998 |
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10040315 |
Oct 29, 2001 |
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09103754 |
Jun 24, 1998 |
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6344548 |
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60107771 |
Nov 9, 1998 |
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Current U.S.
Class: |
800/14 ;
424/146.1 |
Current CPC
Class: |
C12N 9/1029 20130101;
C12N 15/8247 20130101; C12P 7/6463 20130101; C12P 7/6454 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
800/014 ;
424/146.1 |
International
Class: |
A01K 067/027; A61K
039/395 |
Goverment Interests
[0002] This invention was made with Government support under Grant
Nos. R01 52069 and R01 57170 awarded by the National Institute of
Health and support from the Veteran's Administration. The
Government has certain rights in this invention.
Claims
What is claimed is:
1. A non-human animal model characterized by having abnormal DGAT
activity, wherein said abnormal DGAT activity results from a DGAT
genomic modification.
2. The animal model according to claim 1, wherein the animal is
further characterized by having decreased endogenous DGAT
expression relative to a corresponding wild-type control.
3. The animal according to claim 2, wherein the animal is
heterozygous for a defect in an endogenous DGAT gene.
4. The animal according to claims 2, wherein the animal is
homozygous for a defect in an endognenous DGAT gene.
5. The animal according to claim 4, wherein said animal is an
endogenous DGAT gene knockout animal.
6. The animal according to claim 5, wherein said animal further
comprises an exogenous DGAT coding sequence which is expressed in
said animal.
7. The animal according to claim 6, wherein said exogenous DGAT
coding sequence is a human DGAT coding sequence.
8. The animal according to claim 1, wherein the animal is further
characterized by having increased endogenous DGAT expression
relative to a corresponding wild-type control.
9. The animal according to claim 8, wherein said increased
endogenous DGAT expression results from the presence of extra
endogenous DGAT coding sequences.
10. A cell having a disrupted endogenous DGAT locus.
11. The cell according to claim 10, wherein said cell is an
endogenous DGAT knockout.
12. The cell according to claim 11, wherein said cell is a
non-human cell.
13. The cell according to claim 12, wherein said cell is a mouse
cell.
14. The cell according to claim 13, wherein said cell further
comprises a coding sequence for a human DGAT polypeptide, wherein
said coding sequence is expressed in said cell.
15. A screening assay for determining a candidate agent's DGAT
modulatory activity, said method comprising: (a) contacting a DGAT
polypeptide with said candidate agent; and (b) detecting any change
in activity of said DGAT polypeptide compared to a control to
determine said candidate agent's DGAT modulatory activity.
16. The screening assay according to claim 15, wherein said DGAT
modulatory activity is inhibitory activity.
17. The screening assay according to claim 16, wherein said DGAT
polypeptide is a human DGAT.
18. The screening assay according to claim 16, wherein said DGAT
polypeptide is mouse DGAT.
19. The screening assay according to claim 16, wherein said
screening assay is an in vitro screening assay.
20. The screening assay according to claim 16, wherein said
screening assay is an in vivo screening assay.
21. The screening assay according to claim 20, wherein said
contacting comprises introducing said candidate agent into a cell
that includes said DGAT polypeptide.
22. The screening assay according to claim 21, wherein said cell is
a cell according to claim 14.
23. The screening assay according to claim 21, wherein said
contacting comprises administering said candidate agent to an
animal according to claim 1.
24. A screening assay for determining a candidate agent's DGAT
expression modulatory activity, said assay comprising: (a)
contacting a DGAT polypeptide expression cassette with said
candidate agent; and (b) detecting any change in expression of said
DGAT polypeptide expression cassette compared to a control to
determine said candidate agent's DGAT expression modulatory
activity.
25. The screening assay according to claim 24, wherein said
expression modulatory activity is inhibitory activity.
26. The screening assay according to claim 24, wherein assay is in
vitro.
27. The screening assay according to claim 24, wherein said assay
is in vivo.
28. The screening assay according to claim 24, wherein said DGAT
polypeptide is a human DGAT.
29. The screening assay according to claim 24, wherein said DGAT
polypeptide is a mouse DGAT.
30. A non-human polypeptide having DGAT activity present in other
than its naturally occurring environment, wherein when said
polypeptide has the amino acid sequence of a naturally occurring
protein it is substantially free of any of its constituents of its
naturally occurring environment.
31. The polypeptide according to claim 30, wherein said polypeptide
has an amino acid sequence of a naturally occurring DGAT
protein.
32. The polypeptide according to claim 31, wherein said naturally
occurring DGAT-protein is an animal DGAT protein.
33. The polypeptide according to claim 32, wherein said animal DGAT
protein is a mammalian DGAT protein.
34. The polypeptide according to claim 33, wherein said DGAT
protein is a mouse protein.
35. The polypeptide according to claim 34, wherein said mouse DGAT
protein comprises SEQ ID NO:07.
36. Substantially pure mammalian non-human DGAT.
37. Isolated mammalian non-human DGAT.
38. A fragment of a polypeptide according to claim 30.
39. A monoclonal antibody binding specifically to a polypeptide
having DGAT activity.
40. The monoclonal antibody according to claim 39, wherein said
antibody inhibits DGAT activity of said polypeptide.
41. A method for inhibiting the activity of a DGAT protein, said
method comprising: contacting said DGAT protein with an agent that
inhibits the activity of said DGAT protein.
42. The method according to claim 41, wherein said agent is a small
molecule.
43. The method according to claim 42, wherein said agent is an
antibody.
44. The method according to claim 42, wherein said agent is a
monoclonal antibody.
45. A method of modulating a symptom in a mammalian host of a
disease condition associated with DGAT activity, said method
comprising: administering to said host a pharmaceutical composition
comprising an effective amount of an active agent that modulates
said DGAT activity in said host.
46. The method according to claim 45, wherein said symptom is
hypertriglycemia.
47. The method according to claim 45, wherein said syptom is
obesity
48. A plant polynucleotide present in other than its natural
environment encoding a product having DGAT activity.
49. The polynucleotide according to claim 48, wherein said
polynucleotide comprises a sequence substantially similar or
identical to SEQ ID NO: 04.
50. A nucleic acid that hybridizes under stringent conditions to a
nucleic acid consisting of SEQ ID NO:04.
51. A plant polypeptide having DGAT activity present in other than
its naturally occurring environment, wherein when said polypeptide
has the amino acid sequence of a naturally occurring protein it is
substantially free of any of its constituents of its naturally
occurring environment.
52. The polypeptide according to claim 51, wherein said polypeptide
has an amino acid sequence of a naturally occurring DGAT
protein.
53. The polypeptide according to claim 52, wherein said plant DGAT
protein comprises a sequence encoded by a polynucleotide comprising
the sequence of SEQ ID NO:04.
54. A fragment of a polypeptide according to claim 51.
55. An expression cassette comprising a transcriptional initiation
region functional in an expression host, a polynucleotide having a
nucleotide sequence found in the nucleic acid according to claim 48
under the transcriptional regulation of said transcriptional
initiation region, and a transcriptional termination region
functional in said expression host.
56. A cell, or the progeny thereof, comprising an expression
cassette according to claim 55 as part of an extrachromosomal
element or integrated into the genome of a host cell as a result of
introduction of said expression cassette into said host cell.
57. A method of producing a polypeptide having plant DGAT activity,
said method comprising: growing a cell according to claim 56,
whereby said polypeptide is expressed; and isolating said
polypeptide substantially free of other proteins.
58. An antibody binding specifically to a polypeptide having plant
DGAT activity.
59. A method of producing a triacylglycerol, said method
comprising: contacting a diacylglyercol and fatty acyl CoA with a
plant DGAT polypeptide under conditions sufficient to said
triacylglycerol to be produced.
60. A DGAT transgenic plant.
61. The transgenic plant according to claim 60, wherein said plant
is capable of producing seeds higher in oil content than the
corresponding wild-type.
62. The seeds produced by the plant according to claim 61.
63. A method of producing an oil seed having a higher oil content
as compared to wild-type, said method comprising: growing a DGAT
transgenic plant according to claim 61; and harvesting seeds from
said DGAT transgenic plant.
64. In a method of producing oil from seeds, the improvement
comprising: producing oil from the seeds produced according to the
method of claim 63.
65. In a method of identifying a plant DGAT polynucleotide, the
improvement comprising: employing a probe comprising a sequence
substantially similar or identical to SEQ ID NO:04 to identify said
plant DGAT polynucleotide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/339,472 filed on Jun. 23, 1999; which application
claims priority to the filing date of U.S. Provisional Patent
Application Serial No. 60/107,771 filed Nov. 9, 1998, the
disclosure of which is herein incorporated by reference; and which
application is also a continuation-in-part of PCT application
serial no. PCT/US98/17883, filed Aug. 28, 1998; and a continuation
in part of application Ser. No. 09/103,754, filed Jun. 24, 1998;
this disclosures of which applications are herein incorporated by
reference.
INTRODUCTION
[0003] 1. Field of the Invention
[0004] The field of the invention is enzymes, particularly
acyltransferases.
[0005] 2. Background of the Invention
[0006] Diacylglycerol O-Acyltransferase (EC 2.3.1.20), also known
as diglyceride acyltransferase or DGAT, is a critical enzyme in
triacylglycerol synthesis. Triacylglycerols are quantitatively the
most important storage form of energy for eukaryotic cells. DGAT
catalyzes the rate-limiting and terminal step in triacylglycerol
synthesis using diacylglycerol and fatty acyl CoA as substrates. As
such, DGAT plays a fundamental role in the metabolism of cellular
diacylglycerol and is important in higher eukaryotes for intestinal
fat absorption, lipoprotein assembly, fat storage in adipocytes,
milk production and possibly egg production and sperm
maturation.
[0007] Because of its central role in a variety of different
processes, there is much interest in the identification of
polynucleotides encoding proteins having DGAT activity, as well as
the proteins encoded thereby.
[0008] Relevant Literature
[0009] References of interest include: U.S. Pat. No. 6,100,077;
Cases et al., the FASEB Journal (Mar. 20, 1998) Vol. 12.,
No.(5):A814; Cases et al., Proc. Natl. Acad. Sci. USA (October
1998) 95:13018-13023; and Genbank Accession No.AF078752 (Nov. 11,
1998); Genbank Accession No. AAC63997 (Oct. 15, 1998); and Genbank
Accession No. AF059202 (Oct. 15, 1998).
SUMMARY OF THE INVENTION
[0010] Nucleic acid compositions encoding polypeptide products with
diglyceride acyltransferase activity, as well as the polypeptide
products encoded thereby and methods for producing the same, are
provided. Also provided are: methods and compositions for
modulating DGAT activity; DGAT transgenic cells, animals and
plants, as well as methods for their preparation; and methods for
making triglycerides and triglyceride compositions, as well as the
compositions produced by these methods. The subject methods and
compositions find use in a variety of different applications,
including research, medicine, agriculture and industry.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 depicts the strategy employed to generate the DGAT
knockout mice of the subject invention.
[0012] FIG. 2 shows the DGAT activity in various tissue membranes
of DGAT knockout and wild type mice.
[0013] FIG. 3 compares the fat pads of wild type and DGAT knockout
mice.
[0014] FIG. 4 compares the body weights of wild-type and DGAT
knockout mice fed a chow diet.
[0015] FIG. 5 compares the body weights of wild type and DGAT
knockout mice fed a high fat diet.
[0016] FIGS. 6A and 6B compare the body weight and fat pads of 30
week old female wild type and DGAT knockout mice fed a high fat
diet.
[0017] FIG. 7 compares the glucose and free fatty acid levels in
wild type and DGAT knockout mice.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Nucleic acid compositions encoding polypeptide products with
diglyceride acyltransferase activity, as well as the polypeptide
products encoded thereby and methods for producing the same, are
provided. Also provided are: methods and compositions for
modulating DGAT activity, e.g. in the treatment of disease
conditions associated with DGAT activity, including obesity; DGAT
transgenic cells, animals, plants and fungi, and methods for their
preparation, e.g. for use in research, food production, industrial
feedstock production, etc.; and methods for making triglycerides
and triglyceride compositions, e.g. oils. The methods and
compositions of the subject invention find use in a variety of
different applications and fields, including research, medicine,
agriculture and industry.
[0019] Before the subject invention is described further, it is to
be understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope
of the appended claims. It is also to be understood that the
terminology employed is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
[0020] In this specification and the appended claims, the singular
forms "a," "an" and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0021] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0023] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the subject
components of the invention that are described in the publications,
which components might be used in connection with the presently
described invention.
[0024] Nucleic Acid Compositions
[0025] Nucleic acid compositions encoding polypeptide products, as
well as fragments thereof, having diglyceride acetyltransferase
(DGAT) activity are provided. By nucleic acid composition is meant
a composition comprising a sequence of DNA having an open reading
frame that encodes a DGAT polypeptide, i.e. a gene encoding a
polypeptide having DGAT activity, and is capable, under appropriate
conditions, of being expressed as a DGAT polypeptide. Also
encompassed in this term are nucleic acids that are homologous or
substantially similar or identical to the nucleic acids encoding
DGAT polypeptides or proteins. Thus, the subject invention provides
genes encoding mammalian DGAT, such as genes encoding human DGAT
and homologs thereof and mouse DGAT and homologs thereof, as well
as plant DGAT, such as Arabidopsis thaliana DGAT and homologs
thereof. In other words, both animal and plant genes encoding DGAT
proteins are provided by the subject invention. In certain
embodiments, non-human nucleic acids encoding DGAT products are
preferred. The coding sequence of the human DGAT gene, i.e. the
human cDNA encoding the human DGAT enzyme, includes or comprises a
nucleic acid sequence substantially the same as or identical to
that identified as SEQ ID NO:01 or SEQ ID NO:02, infra. A nucleic
acid sequence encoding the full length human DGAT enzyme (i.e. a
protein having or including the sequence of SEQ ID NO:05) is also
of interest. Also of interest is a nucleic acid encoding the full
length sequence of the human DGAT protein, as shown in SEQ ID
NO:06. The coding sequence of the mouse DGAT gene, i.e. the mouse
cDNA encoding the mouse DGAT enzyme, has the nucleic acid sequence
identified as SEQ ID NO:03, infra. Also of interest is the sequence
identified as SEQ ID NO:09. The coding sequence of the A. thaliana
DGAT gene, i.e. the A. thaliana cDNA encoding the A. thaliana DGAT
enzyme, comprises or includes the nucleic acid sequence identified
as SEQ ID NO:04, infra.
[0026] The source of homologous genes to those specifically listed
above may be any species, including both animal and plant species,
e.g., primate species, particularly human; rodents, such as rats
and mice, canines, felines, bovines, ovines, equines, yeast,
nematodes, etc. Between mammalian species, e.g., human and mouse,
homologs have substantial sequence similarity, e.g. at least 75%
sequence identity, usually at least 90%, more usually at least 95%
between nucleotide sequences. Sequence similarity is calculated
based on a reference sequence, which may be a subset of a larger
sequence, such as a conserved motif, coding region, flanking
region, etc. A reference sequence will usually be at least about 18
nt long, more usually at least about 30 nt long, and may extend to
the complete sequence that is being compared. Algorithms for
sequence analysis are known in the art, such as BLAST, described in
Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless specified
otherwise, all sequence identity values provided herein are
determined using GCG (Genetics Computer Group, Wisconsin Package,
Standard Settings, gap creation penalty 3.0, gap extension penalty
0.1). The sequences provided herein are essential for recognizing
DGAT-related and homologous polynucleotides in database
searches.
[0027] Nucleic acids encoding the DGAT proteins and DGAT
polypeptides of the subject invention may be cDNAs or genomic DNAs,
as well as fragments thereof. The term "DGAT-gene" shall be
intended to mean the open reading frame encoding specific DGAT
proteins and polypeptides, and DGAT introns, as well as adjacent 5'
and 3' non-coding nucleotide sequences involved in the regulation
of expression, up to about 20 kb beyond the coding region, but
possibly further in either direction. The gene may be introduced
into an appropriate vector for extrachromosomal maintenance or for
integration into a host genome.
[0028] The term "cDNA" as used herein is intended to include all
nucleic acids that share the arrangement of sequence elements found
in native mature mRNA species, where sequence elements are exons
and 3' and 5' non-coding regions. Normally mRNA species have
contiguous exons, with the intervening introns, when present, being
removed by nuclear RNA splicing, to create a continuous open
reading frame encoding a DGAT protein.
[0029] A genomic sequence of interest comprises the nucleic acid
present between the initiation codon and the stop codon, as defined
in the listed sequences, including all of the introns that are
normally present in a native chromosome. It may further include the
3' and 5' untranslated regions found in the mature mRNA. It may
further include specific transcriptional and translational
regulatory sequences, such as promoters, enhancers, etc., including
about 1 kb, but possibly more, of flanking genomic DNA at either
the 5' or 3' end of the transcribed region. The genomic DNA may be
isolated as a fragment of 100 kbp or smaller; and substantially
free of flanking chromosomal sequence. The genomic DNA flanking the
coding region, either 3' or 5', or internal regulatory sequences as
sometimes found in introns, contains sequences required for proper
tissue and stage specific expression.
[0030] The nucleic acid compositions of the subject invention may
encode all or a part of the subject DGAT proteins and polypeptides,
described in greater detail infra. Double or single stranded
fragments may be obtained from the DNA sequence by chemically
synthesizing oligonucleotides in accordance with conventional
methods, by restriction enzyme digestion, by PCR amplification,
etc. For the most part, DNA fragments will be of at least 15 nt,
usually at least 18 nt or 25 nt, and may be at least about 50
nt.
[0031] The DGAT-genes of the subject invention are isolated and
obtained in substantial purity, generally as other than an intact
chromosome. Usually, the DNA will be obtained substantially free of
other nucleic acid sequences that do not include a DGAT sequence or
fragment thereof, generally being at least about 50%, usually at
least about 90% pure and are typically "recombinant", i.e. flanked
by one or more nucleotides with which it is not normally associated
on a naturally occurring chromosome.
[0032] Also provided are nucleic acids that hybridize to the above
described nucleic acids under stringent conditions. An example of
stringent hybridization conditions is hybridization at 50.degree.
C. or higher and 0.1.times.SSC (15 mM sodium chloride/1.5 mM sodium
citrate). Another example of stringent hybridization conditions is
overnight incubation at 42.degree. C. in a solution: 50% formamide,
5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C. Stringent hybridization conditions are hybridization
conditions that are at least as stringent as the above
representative conditions. Other stringent hybridization conditions
are known in the art and may also be employed to identify nucleic
acids of this particular embodiment of the invention.
[0033] Also provided are nucleic acids that encode the proteins
encoded by the above described nucleic acids, but differ in
sequence from the above described nucleic acids due to the
degeneracy of the genetic code.
[0034] In addition to the plurality of uses described in greater
detail in following sections, the subject nucleic acid compositions
find use in the preparation of all or a portion of the DGAT
polypeptides, as described below.
[0035] Polypeptide Compositions
[0036] Also provided by the subject invention are polypeptides
having DGAT activity, i.e. capable of catalyzing the acylation of
diacylglycerol. In addition to being capable of catalyzing the
esterification of diacylglycerol with a fatty acyl CoA substrates,
the subject proteins are incapable of esterifying, at least to any
substantial extent, the following substrates: cholesterol,
25-hydroxy-, 27-hydroxy-, 7-hydroxy- or 7-hydroxycholesterols,
7-ketocholesterol, vitamins D2 and D3, vitamin E,
dehydrepiandrosterone, retinol, ethanol, sitosterol, lanosterol and
ergosterol.
[0037] The term polyeptide composition as used herein refers to
both the full length proteins as well as portions or fragments
thereof. Also included in this term are variations of the naturally
occurring proteins, where such variations are homologous or
substantially similar to the naturally occurring protein, as
described in greater detail below, be the naturally occurring
protein the human protein, mouse protein, or protein from some
other species which naturally expresses a DGAT enzyme, be that
species animal or plant. In the following description of the
subject invention, the term DGAT is used to refer not only to the
human form of the enzyme, but also to homologs thereof expressed in
non-human species, including plant species.
[0038] The subject DGAT proteins are, in their natural environment,
trans-membrane proteins. The subject proteins are characterized by
the presence of at least one potential N-linked glycosylation site,
at least one potential tyrosine phosphorylation site, and multiple
hydrophobic domains, including 6 to 12 hydrophobic domains capable
of serving as trans-membrane regions. The proteins range in length
from about 400 to 650, usually from about 475 to 525 and more
usually from about 485 to 500 amino acid residues, and the
projected molecular weight of the subject proteins based solely on
the number of amino acid residues in the protein ranges from about
50 to 80, usually from about 55 to 75 and more usually from about
60 to 65 kDa, where the actual molecular weight may vary depending
on the amount of glycolsylation of the protein and the apparent
molecular weight may be considerably less (e.g. 40 to 50 kDa)
because of SDS binding on gels.
[0039] The amino acid sequences of the subject proteins are
characterized by having at least some homology to a corresponding
ACAT protein from the same species, e.g. a human DGAT protein has
at least some sequence homology with the human ACAT-1 protein, the
mouse DGAT protein has at least some sequence homology with the
mouse ACAT-1 protein, etc., where the sequence homology will not
exceed about 50%, and usually will not exceed about 40% and more
usually will not exceed about 25%, but will be at least about 15%
and more usually at least about 20%, as determined using GCG
(Genetics Computer Group, Wisconsin Package, Standard Settings, Gap
Creation Penalty 3.0, Gap Extension Penalty 0.1).
[0040] Of particular interest in many embodiments are proteins that
are non-naturally glycosylated. By non-naturally glycosylated is
meant that the protein has a glycosylation pattern, if present,
which is not the same as the glycosylation pattern found in the
corresponding naturally occurring protein. For example, human DGAT
of the subject invention and of this particular embodiment is
characterized by having a glycosylation pattern, if it is
glycosylated at all, that differs from that of naturally occurring
human DGAT. Thus, the non-naturally glycosylated DGAT proteins of
this embodiment include non-glycosylated DGAT proteins, i.e.
proteins having no covalently bound glycosyl groups.
[0041] Of particular interest in certain embodiments is the human
DGAT protein, where the human DGAT protein of the subject invention
has an amino acid sequence that comprises or includes a region
substantially the same as or identical to the sequence appearing as
SEQ ID NO:05 infra. The sequence of the full length human DGAT
protein includes the sequence provided in SEQ ID NO:06 infra. As
such, DGAT proteins having an amino acid sequence that is
substantially the same as or identical to the sequence of SEQ ID
NO:6 are of interest. By substantially the same as is meant a
protein having a region with a sequence that has at least about
75%, usually at least about 90% and more usually at least about 98%
sequence identity with the sequence of SED ID NO:06, as measured by
GCG, supra. Of particular interest in other embodiments is the
mouse DGAT protein, where the mouse DGAT protein of the subject
invention has an amino acid sequence that is substantially the same
as or identical to the sequence appearing as SEQ ID NO:07, infra.
Also of interest is SEQ ID NO:10. Also of particular interest in
yet other embodiments of the subject invention is the A. thaliana
DGAT protein, where the A. thaliana DGAT protein of the subject
invention has an amino acid sequence encoded by the nucleic acid
comprising the sequence appearing as SEQ ID NO:04, infra.
[0042] In addition to the specific DGAT proteins described above,
homologs or proteins (or fragments thereof) from other species,
i.e. other animal or plant species, are also provided, where such
homologs or proteins may be from a variety of different types of
species, including animals, such as mammals, e.g. rodents, such as
mice, rats; domestic animals, e.g. horse, cow, dog, cat; and
humans, as well as non-mammalian species, e.g. avian, insect and
the like, as well plant species. By homolog is meant a protein
having at least about 35%, usually at least about 40% and more
usually at least about 60% amino acid sequence identity the
specific DGAT proteins as identified in SEQ ID NOS: 04 to 06, where
sequence identity is determined using GCG, supra.
[0043] The DGAT proteins of the subject invention (e.g. human DGAT
or a homolog thereof; non-human DGAT proteins, e.g. mouse DGAT,
plant DGAT) are present in a non-naturally occurring environment,
e.g. are separated from their naturally occurring environment. In
certain embodiments, the subject DGAT is present in a composition
that is enriched for DGAT as compared to DGAT in its naturally
occurring environment. As such, purified DGAT is provided, where by
purified is meant that DGAT is present in a composition that is
substantially free of non DGAT proteins, where by substantially
free is meant that less than 90%, usually less than 60% and more
usually less than 50% of the composition is made up of non-DGAT
proteins. For compositions that are enriched for DGAT proteins,
such compositions will exhibit a DGAT activity of at least about
100, usually at least about 200 and more usually at least about
1000 pmol triglycerides formed/mg protein/min, where such activity
is determined by the assay described in the Experimental Section,
infra.
[0044] In certain embodiments of interest, the DGAT protein is
present in a composition that is substantially free of the
constituents that are present in its naturally occurring
environment. For example, a human DGAT protein comprising
composition according to the subject invention in this embodiment
will be substantially, if not completely, free of those other
biological constituents, such as proteins, carbohydrates, lipids,
etc., with which it is present in its natural environment. As such,
protein compositions of these embodiments will necessarily differ
from those that are prepared by purifying the protein from a
naturally occurring source, where at least trace amounts of the
protein's constituents will still be present in the composition
prepared from the naturally occurring source.
[0045] The DGAT of the subject invention may also be present as an
isolate, by which is meant that the DGAT is substantially free of
both non-DGAT proteins and other naturally occurring biologic
molecules, such as oligosaccharides, polynucleotides and fragments
thereof, and the like, where substantially free in this instance
means that less than 70%, usually less than 60% and more usually
less than 50% of the composition containing the isolated DGAT is a
non-DGAT naturally occurring biological molecule. In certain
embodiments, the DGAT is present in substantially pure form, where
by substantially pure form is meant at least 95%, usually at least
97% and more usually at least 99% pure.
[0046] In addition to the naturally occurring DGAT proteins, DGAT
polypeptides which vary from the naturally occurring DGAT proteins
are also provided. By DGAT polypeptides is meant proteins having an
amino acid sequence encoded by an open reading frame (ORF) of a
DGAT gene, described supra, including the full length DGAT protein
and fragments thereof, particularly biologically active fragments
and/or fragments corresponding to functional domains; and including
fusions of the subject polypeptides to other proteins or parts
thereof. Fragments of interest will typically be at least about 10
aa in length, usually at least about 50 aa in length, and may be as
long as 300 aa in length or longer, but will usually not exceed
about 1000 aa in length, where the fragment will have a stretch of
amino acids that is identical to a DGAT protein of SEQ ID NO:5, SEQ
ID NO:06, SEQ ID NO:07 or a homolog thereof; of at least about 10
aa, and usually at least about 15 aa, and in many embodiments at
least about 50 aa in length.
[0047] Preparation of DGAT Polypeptides
[0048] The subject DGAT proteins and polypeptides may be obtained
from naturally occurring sources, but are preferably synthetically
produced. Where obtained from naturally occurring sources, the
source chosen will generally depend on the species from which the
DGAT is to be derived.
[0049] The subject DGAT polypeptide compositions may be
synthetically derived by expressing a recombinant gene encoding
DGAT, such as the polynucleotide compositions described above, in a
suitable host. For expression, an expression cassette may be
employed. The expression vector will provide a transcriptional and
translational initiation region, which may be inducible or
constitutive, where the coding region is operably linked under the
transcriptional control of the transcriptional initiation region,
and a transcriptional and translational termination region. These
control regions may be native to a DGAT gene, or may be derived
from exogenous sources.
[0050] Expression vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences encoding heterologous proteins.
A selectable marker operative in the expression host may be
present. Expression vectors may be used for the production of
fusion proteins, where the exogenous fusion peptide provides
additional functionality, i.e. increased protein synthesis,
stability, reactivity with defined antisera, an enzyme marker, e.g.
-galactosidase, etc.
[0051] Expression cassettes may be prepared comprising a
transcription initiation region, the gene or fragment thereof, and
a transcriptional termination region. Of particular interest is the
use of sequences that allow for the expression of functional
epitopes or domains, usually at least about 8 amino acids in
length, more usually at least about 15 amino acids in length, to
about 25 amino acids, and up to the complete open reading frame of
the gene. After introduction of the DNA, the cells containing the
construct may be selected by means of a selectable marker, the
cells expanded and then used for expression.
[0052] DGAT proteins and polypeptides may be expressed in
prokaryotes or eukaryotes in accordance with conventional ways,
depending upon the purpose for expression. For large scale
production of the protein, a unicellular organism, such as E. coli,
B. subtilis, S. cerevisiae, insect cells in combination with
baculovirus vectors, or cells of a higher organism such as
vertebrates, particularly mammals, e.g. COS 7 cells, may be used as
the expression host cells. In some situations, it is desirable to
express the DGAT gene in eukaryotic cells, where the DGAT protein
will benefit from native folding and post-translational
modifications. Small peptides can also be synthesized in the
laboratory. Polypeptides that are subsets of the complete DGAT
sequence may be used to identify and investigate parts of the
protein important for function.
[0053] Specific expression systems of interest include bacterial,
yeast, insect cell and mammalian cell derived expression systems.
Representative systems from each of these categories is are
provided below:
[0054] Bacteria. Expression systems in bacteria include those
described in Chang et al., Nature (1978) 275:615; Goeddel et al.,
Nature (1979) 281:544; Goeddel et al., Nucleic Acids Res. (1980)
8:4057; EP 0 036,776; U.S. Pat. No. 4,551,433; DeBoer et al., Proc.
Natl. Acad. Sci. (USA) (1983) 80:21-25; and Siebenlist et al., Cell
(1980) 20:269.
[0055] Yeast. Expression systems in yeast include those described
in Hinnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito
et al., J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell.
Biol. (1986) 6:142; Kunze et al., J. Basic Microbiol. (1985)
25:141; Gleeson et al., J. Gen. Microbiol. (1986) 132:3459;
Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302; Das et al., J.
Bacteriol. (1984) 158:1165; De Louvencourt et al., J. Bacteriol.
(1983) 154:737; Van den Berg et al., Bio/Technology (1990) 8:135;
Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg et al., Mol.
Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555;
Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.
Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;
Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;
Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl.
Acad. Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J.
(1985) 4:475479; EP 0 244,234; and WO 91/00357.
[0056] Insect Cells. Expression of heterologous genes in insects is
accomplished as described in U.S. Pat. No. 4,745,051; Friesen et
al., "The Regulation of Baculovirus Gene Expression", in: The
Molecular Biology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0
127,839; EP 0 155,476; and Vlak et al., J. Gen. Virol. (1988)
69:765-776; Miller et al., Ann. Rev. Microbiol. (1988) 42:177;
Carbonell et al., Gene (1988) 73:409; Maeda et al., Nature (1985)
315:592-594; Lebacq-Verheyden et al., Mol. Cell. Biol. (1988)
8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8844;
Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988)
7:99. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts are described in Luckow et
al., Bio/Technology (1988) 6:47-55, Miller et al., Generic
Engineering (1986) 8:277-279, and Maeda et al., Nature (1985)
315:592-594.
[0057] Mammalian Cells. Mammalian expression is accomplished as
described in Dijkema et al., EMBO J. (1985) 4:761, Gorman et al.,
Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell
(1985) 41:521 and U.S. Pat. No. 4,399,216. Other features of
mammalian expression are facilitated as described in Ham and
Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem.
(1980) 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,
4,560,655, WO 90/103430, WO 87/00195, and U.S. RE Pat. No.
30,985.
[0058] When any of the above host cells, or other appropriate host
cells or organisms, are used to replicate and/or express the
polynucleotides or nucleic acids of the invention, the resulting
replicated nucleic acid, RNA, expressed protein or polypeptide, is
within the scope of the invention as a product of the host cell or
organism.
[0059] Once the source of the protein is identified and/or
prepared, e.g. a transfected host expressing the protein is
prepared, the protein is then purified to produce the desired DGAT
comprising composition. Any convenient protein purification
procedures may be employed, where suitable protein purification
methodologies are described in Guide to Protein Purification,
(Deuthser ed.) (Academic Press, 1990). For example, a lysate may
prepared from the original source, e.g. naturally occurring cells
or tissues that express DGAT or the expression host expressing
DGAT, and purified using HPLC, exclusion chromatography, gel
electrophoresis, affinity chromatography, and the like.
[0060] Once the gene corresponding to a selected polynucleotide is
identified, its expression can be regulated in the cell to which
the gene is native. For example, an endogenous gene of a cell can
be regulated by an exogenous regulatory sequence as disclosed in
U.S. Pat. No. 5,641,670; the disclosure of which is herein
incorporated by reference.
[0061] Methods and Compositions Having Research Application
[0062] Also provided by the subject invention are methods and
compositions having research applications, such as in the study of
the acylglycerol metabolism, in the identification of key
components of the triglyceride synthesis pathway, in the
identification of triglyceride synthesis modulatory agents, e.g.
DGAT inhibitors or enhancers, and the like.
[0063] The subject nucleic acid compositions find use in a variety
of research applications. Research applications of interest
include: the identification of DGAT homologs; as a source of novel
promoter elements; the identification of DGAT expression regulatory
factors; as probes and primers in hybridization applications, e.g.
PCR; the identification of expression patterns in biological
specimens; the preparation of cell or animal models for DGAT
function; the preparation of in vitro models for DGAT function;
etc.
[0064] Homologs of DGAT are identified by any of a number of
methods. A fragment of the provided cDNA may be used as a
hybridization probe against a cDNA library from the target organism
of interest, where low stringency conditions are used. The probe
may be a large fragment, or one or more short degenerate primers.
Nucleic acids having sequence similarity are detected by
hybridization under low stringency conditions, for example, at
50.degree. C. and 6.times.SSC (0.9 M sodium chloride/0.09 M sodium
citrate) and remain bound when subjected to washing at 55.degree.
C. in 1.times.SSC (0.15 M sodium chloride/0.015 M sodium citrate).
Sequence identity may be determined by hybridization under
stringent conditions, for example, at 50.degree. C. or higher and
0.1.times.SSC (15 mM sodium chloride/01.5 mM sodium citrate).
Nucleic acids having a region of substantial identity to the
provided DGAT sequences, e.g. allelic variants, genetically altered
versions of the gene, etc., bind to the provided DGAT sequences
under stringent hybridization conditions. By using probes,
particularly labeled probes of DNA sequences, one can isolate
homologous or related genes. One can also use sequence information
derived from the polynucleotide compositions of the subject
invention to prepare electronic "probes" for use in searching of
computer based sequence date, e.g. BLAST searches EST
databases.
[0065] The sequence of the 5' flanking region of the subject
nucleic acid compositions may be utilized as a source for promoter
elements, including enhancer binding sites, that provide for
developmental regulation in tissues where DGAT is expressed. The
tissue specific expression is useful for determining the pattern of
expression, and for providing promoters that mimic the native
pattern of expression. Naturally occurring polymorphisms in the
promoter region are useful for determining natural variations in
expression, particularly those that may be associated with
disease.
[0066] Alternatively, mutations may be introduced into the promoter
region to determine the effect of altering expression in
experimentally defined systems. Methods for the identification of
specific DNA motifs involved in the binding of transcriptional
factors are known in the art, e.g. sequence similarity to known
binding motifs, gel retardation studies, etc. For examples, see
Blackwell et al. (1995), Mol. Med. 1:194-205; Mortlock et al.
(1996), Genome Res. 6:327-33; and Joulin and Richard-Foy (1995),
Eur. J. Biochem. 232:620-626.
[0067] The regulatory sequences may be used to identify cis acting
sequences required for transcriptional or translational regulation
of DGAT gene expression, especially in different tissues or stages
of development, and to identify cis acting sequences and
trans-acting factors that regulate or mediate DGAT gene expression.
Such transcription or translational control regions may be operably
linked to a DGAT gene in order to promote expression of wild type
or altered DGAT or other proteins of interest in cultured cells, or
in embryonic, fetal or adult tissues, and for gene therapy.
[0068] Small DNA fragments are useful as primers for PCR,
hybridization screening probes, etc. Larger DNA fragments, i.e.
greater than 100 nt are useful for production of the encoded
polypeptide, as described in the previous section. For use in
amplification reactions, such as PCR, a pair of primers will be
used. The exact composition of the primer sequences is not critical
to the invention, but for most applications the primers will
hybridize to the subject sequence under stringent conditions, as
known in the art. It is preferable to choose a pair of primers that
will generate an amplification product of at least about 50 nt,
preferably at least about 100 nt. Algorithms for the selection of
primer sequences are generally known, and are available in
commercial software packages. Amplification primers hybridize to
complementary strands of DNA, and will prime towards each
other.
[0069] The DNA may also be used to identify expression of the gene
in a biological specimen. The manner in which one probes cells for
the presence of particular nucleotide sequences, as genomic DNA or
RNA, is well established in the literature. Briefly, DNA or mRNA is
isolated from a cell sample. The mRNA may be amplified by RT-PCR,
using reverse transcriptase to form a complementary DNA strand,
followed by polymerase chain reaction amplification using primers
specific for the subject DNA sequences. Alternatively, the mRNA
sample is separated by gel electrophoresis, transferred to a
suitable support, e.g. nitrocellulose, nylon, etc., and then probed
with a fragment of the subject DNA as a probe. Other techniques,
such as oligonucleotide ligation assays, in situ hybridizations,
and hybridization to DNA probes arrayed on a solid chip may also
find use. Detection of mRNA hybridizing to the subject sequence is
indicative of DGAT gene expression in the sample.
[0070] The sequence of a DGAT gene, including flanking promoter
regions and coding regions, may be mutated in various ways known in
the art to generate targeted changes in promoter strength, sequence
of the encoded protein, etc. The DNA sequence or protein product of
such a mutation will usually be substantially similar to the
sequences provided herein, i.e. will differ by at least one
nucleotide or amino acid, respectively, and may differ by at least
two but not more than about ten nucleotides or amino acids. The
sequence changes may be substitutions, insertions, deletions, or a
combination thereof. Deletions may further include larger changes,
such as deletions of a domain or exon. Other modifications of
interest include epitope tagging, e.g. with the FLAG system, HA,
etc. For studies of subcellular localization, fusion proteins with
green fluorescent proteins (GFP) may be used.
[0071] Techniques for in vitro mutagenesis of cloned genes are
known. Examples of protocols for site specific mutagenesis may be
found in Gustin et al. (1993), Biotechniques 14:22; Barany (1985),
Gene 37:111-23; Colicelli et al. (1985), Mol. Gen. Genet.
199:537-9; and Prentki et al. (1984), Gene 29:303-13. Methods for
site specific mutagenesis can be found in Sambrook et al.,
Molecular Cloning. A Laboratory Manual, CSH Press 1989, pp.
15.3-15.108; Weiner et al. (1993), Gene 126:35-41; Sayers et al.
(1992), Biotechniques 13:592-6; Jones and Winistorfer (1992),
Biotechniques 12:528-30; Barton et al. (1990), Nucleic Acids Res
18:7349-55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67-70;
and Zhu (1989), Anal Biochem 177:120-4. Such mutated genes may be
used to study structure-function relationships of DGAT, or to alter
properties of the protein that affect its function or
regulation.
[0072] The subject nucleic acids can be used to generate transgenic
hosts, e.g. non-human animals, such as mice, cows, rats, pigs etc.,
plants, fungi, or site specific gene modifications in cell lines.
Examples of transgenic hosts (including cells) include hosts in
which the naturally expressed, i.e., endogenous, DGAT gene has been
disrupted, e.g. DGAT knock-outs, as well as hosts in which DGAT
expression has been amplified, e.g. through introduction of
additional DGAT copies, where the copies may be of endogenous DGAT
sequences, through introduction of strong promoter upstream of the
DGAT gene, and the like. As such, transgenic hosts of interest are
hosts that abnormal DGAT activity compared to a wild type control,
where the abnormal DGAT activity results from a DGAT genomic
modification, e.g., a disruption in an endogenous DGAT locus and/or
an introduction of a DGAT coding sequence into genomic DNA. Using
the nucleic acid compositions of the subject invention, standard
protocols known to those of skill in the art may used to produce
such transgenic hosts that have been genetically manipulated with
respect to the DGAT gene, i.e., DGAT transgenic hosts.
[0073] Transgenic animals may be made through homologous
recombination, where the normal DGAT locus is altered, e.g. as in
DGAT knockouts. Alternatively, a nucleic acid construct is randomly
integrated into the genome. Vectors for stable integration include
plasmids, retroviruses and other animal viruses, YACs, and the
like. DNA constructs for homologous recombination will comprise at
least a portion of the DGAT gene native to the species of the host
animal, wherein the gene has the desired genetic modification(s),
and includes regions of homology to the target locus. DNA
constructs for random integration need not include regions of
homology to mediate recombination. Conveniently, markers for
positive and negative selection are included. Methods for
generating cells having targeted gene modifications through
homologous recombination are known in the art. For various
techniques for transfecting mammalian cells, see Keown et al.
(1990), Meth. Enzymol. 185:527-537.
[0074] For embryonic stem (ES) cells, an ES cell line may be
employed, or embryonic cells may be obtained freshly from a host,
e.g. mouse, rat, guinea pig, cow, etc. Such cells are grown on an
appropriate fibroblast-feeder layer or grown in the presence of
leukemia inhibiting factor (LIF). When ES or embryonic cells have
been transformed, they may be used to produce transgenic animals.
After transformation, the cells are plated onto a feeder layer in
an appropriate medium. Cells containing the construct may be
detected by employing a selective medium. After sufficient time for
colonies to grow, they are picked and analyzed for the occurrence
of homologous recombination or integration of the construct. Those
colonies that are positive may then be used for embryo manipulation
and blastocyst injection. Blastocysts are obtained from 4 to 6 week
old superovulated females. The ES cells are trypsinized, and the
modified cells are injected into the blastocoel of the blastocyst.
After injection, the blastocysts are returned to each uterine horn
of pseudopregnant females. Females are then allowed to go to term
and the resulting offspring screened for the construct. By
providing for a different phenotype of the blastocyst and the
genetically modified cells, chimeric progeny can be readily
detected.
[0075] The resultant chimeric animals are screened for the presence
of the modified gene and males and females having the modification
are mated to produce homozygous progeny. If the gene alterations
cause lethality at some pointin development, tissues or organs can
be maintained as allogeneic or congenic grafts or transplants, or
in in vitro culture. The transgenic animals may be any non-human
mammal, such as laboratory animals, domestic animals, etc.
[0076] Transgenic plants may be produced in a similar manner.
Methods of preparing transgenic plant cells and plants are
described in U.S. Pat. Nos. 5,767,367; 5,750,870; 5,739,409;
5,689,049; 5,689,045; 5,674,731; 5,656,466; 5,633,155; 5,629,470;
5,595,896; 5,576,198; 5,538,879; 5,484,956; the disclosures of
which are herein incorporated by reference. Methods of producing
transgenic plants are also reviewed in Plant Biochemistry and
Molecular Biology (eds Lea & Leegood, John Wiley &
Sons)(1993) pp 275-295. In brief, a suitable plant cell or tissue
is harvested, depending on the nature of the plant species. As
such, in certain instances, protoplasts will be isolated, where
such protoplasts may be isolated from a variety of different plant
tissues, e.g. leaf, hypoctyl, root, etc. For protoplast isolation,
the harvested cells are incubated in the presence of cellulases in
order to remove the cell wall, where the exact incubation
conditions vary depending on the type of plant and/or tissue from
which the cell is derived. The resultant protoplasts are then
separated from the resultant cellular debris by sieving and
centrifugation. Instead of using protoplasts, embryogenic explants
comprising somatic cells may be used for preparation of the
transgenic host. Following cell or tissue harvesting, exogenous DNA
of interest is introduced into the plant cells, where a variety of
different techniques are available for such introduction. With
isolated protoplasts, the opportunity arise for introduction via
DNA-mediated gene transfer protocols, including: incubation of the
protoplasts with naked DNA, e.g. plasmids, comprising the exogenous
coding sequence of interest in the presence of polyvalent cations,
e.g. PEG or PLO; and electroporation of the protoplasts in the
presence of naked DNA comprising the exogenous sequence of
interest. Protoplasts that have successfully taken up the exogenous
DNA are then selected, grown into a callus, and ultimately into a
transgenic plant through contact with the appropriate amounts and
ratios of stimulatory factors, e.g. auxins and cytokinins. With
embryogenic explants, a convenient method of introducing the
exogenous DNA in the target somatic cells is through the use of
particle acceleration or "gene-gun" protocols. The resultant
explants are then allowed to grow into chimera plants, cross-bred
and transgenic progeny are obtained. Instead of the naked DNA
approaches described above, another convenient method of producing
transgenic plants is Agrobacterium mediated transformation. With
Agrobacterium mediated transformation, co-integrative or binary
vectors comprising the exogenous DNA are prepared and then
introduced into an appropriate Agrobacterium strain, e.g. A.
tumefaciens. The resultant bacteria are then incubated with
prepared protoplasts or tissue explants, e.g. leaf disks, and a
callus is produced. The callus is then grown under selective
conditions, selected and subjected to growth media to induce root
and shoot growth to ultimately produce a transgenic plant.
[0077] The modified cells, animals or plants are useful in the
study of DGAT function and regulation. For example, a series of
small deletions and/or substitutions may be made in the host's
native DGAT gene to determine the role of different exons in
various physiological processes. Specific constructs of interest
include anti-sense DGAT, which will block DGAT expression,
expression of dominant negative DGAT mutations, and over-expression
of DGAT genes. Where a DGAT sequence is introduced, the introduced
sequence may be either a complete or partial sequence of a DGAT
gene native to the host, or may be a complete or partial DGAT
sequence that is exogenous to the host animal, e.g., a human DGAT
sequence. A detectable marker, such as lac Z may be introduced into
the DGAT locus, where upregulation of DGAT gene expression will
result in an easily detected change in phenotype. One may also
provide for expression of the DGAT gene or variants thereof in
cells or tissues where it is not normally expressed, at levels not
normally present in such cells or tissues, or at abnormal times of
development, e.g., by introducing suitable expression cassettes
(such as ones having a DGAT coding sequence operably linked to a
detectable marker sequence). The transgenic hosts, e.g. animals,
plants, etc., may be used in functional studies, drug screening,
etc., e.g. to determine the effect of a candidate drug on DGAT
activity, to identify drugs that reduce serum triglyceride levels,
etc. For example, one can assay a candidate agent's ability to
modulate DGAT expression by contacting an appropriate expression
cassette with the agent and determining the effect of the agent on
expression.
[0078] The subject polypeptide compositions can be used to produce
in vitro models of triglyceride synthesis, where such models will
consist of the subject DGAT proteins and other components of
triglyceride synthesis, e.g. substrates, such as diacylglycerol or
metabolic precersors thereof, fatty acyl CoAs and the like, other
components of the triacylglycerol synthetase complex, e.g. acyl CoA
ligase, acyl CoA acyltransferase, monoacyl glycerol
acyltransferase, etc.
[0079] Also provided by the subject invention are screening assays
designed to find modulatory agents of DGAT activity, e.g.
inhibitors or enhancers of DGAT activity, as well as the agents
identified thereby, where such agents may find use in a variety of
applications, including as therapeutic agents, as agricultural
chemicals, etc. The screening methods will typically be assays
which provide for qualitative/quantitative measurements of DGAT
activity in the presence of a particular candidate therapeutic
agent. For example, the assay could be an assay which measures the
acylation activity of DGAT in the presence and absence of a
candidate inhibitor agent. The screening method may be an in vitro
or in vivo format, where both formats are readily developed by
those of skill in the art. Depending on the particular method, one
or more of, usually one of, the components of the screening assay
may be labeled, where by labeled is meant that the components
comprise a detectable moiety, e.g. a fluorescent or radioactive
tag, or a member of a signal producing system, e.g. biotin for
binding to an enzyme-streptavidin conjugate in which the enzyme is
capable of converting a substrate to a chromogenic product. Where
in vitro assays are employed, the various components of the in
vitro assay, e.g. the substrate, the donor, the DGAT protein and
the candidate inhibitor, etc. are combined in a assay mixture under
conditions sufficient for DGAT activity to occur, as described in
the experimental section, infra.
[0080] A variety of other reagents may be included in the screening
assay and reaction mixture. These include reagents like salts,
neutral proteins, e.g. albumin, detergents, etc that are used to
facilitate optimal protein-protein binding and/or reduce
non-specific or background interactions. Reagents that improve the
efficiency of the assay, such as protease inhibitors, nuclease
inhibitors, anti-microbial agents, etc. may be used.
[0081] A variety of different candidate agents may be screened by
the above methods. Candidate agents encompass numerous chemical
classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 50
and less than about 2,500 daltons. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0082] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0083] Using the above screening methods, a variety of different
therapeutic agents may be identified. Such agents may target the
enzyme itself, or an expression regulator factor thereof. Such
agents may inhibitors or promoters of DGAT activity, where
inhibitors are those agents that result in at least a reduction of
DGAT activity as compared to a control and enhancers result in at
least an increase in DGAT activity as compared to a control. Such
agents may be find use in a variety of therapeutic applications, as
described in greater detail below.
[0084] Methods and Compositions Having Medical Applications
[0085] The methods and compositions of the subject invention also
have broad ranging applications in a variety of medical
applications, including diagnostic screening, therapeutic
treatments of pathological conditions, in the regulation of DGAT
activity in desirable ways, and the like.
[0086] The subject invention provides methods of screening
individuals for a predisposition to a disease state or the presence
of disease state, where such screening may focus on the presence of
one or more markers, such as a mutated DGAT gene or expression
regulatory element thereof, observed levels of DGAT; the expression
level of the DGAT gene in a biological sample of interest; and the
like.
[0087] Samples, as used herein, include biological fluids such as
blood, cerebrospinal fluid, tears, saliva, lymph, semen, dialysis
fluid and the like; organ or tissue culture derived fluids; and
fluids extracted from physiological tissues. Also included in the
term are derivatives and fractions of such fluids. The cells may be
dissociated, in the case of solid tissues, or tissue sections may
be analyzed. Alternatively a lysate of the cells may be
prepared.
[0088] A number of methods are available for determining the
expression level of a gene or protein in a particular sample.
Diagnosis may be performed by a number of methods to determine the
absence or presence or altered amounts of normal or abnormal DGAT
in a patient sample. For example, detection may utilize staining of
cells or histological sections with labeled antibodies, performed
in accordance with conventional methods. Cells are permeabilized to
stain cytoplasmic molecules. The antibodies of interest are added
to the cell sample, and incubated for a period of time sufficient
to allow binding to the epitope, usually at least about 10 minutes.
The antibody may be labeled with radioisotopes, enzymes,
fluorescers, chemiluminescers, or other labels for direct
detection. Alternatively, a second stage antibody or reagent is
used to amplify the signal. Such reagents are well known in the
art. For example, the primary antibody may be conjugated to biotin,
with horseradish peroxidase-conjugated avidin added as a second
stage reagent. Alternatively, the secondary antibody conjugated to
a flourescent compound, e.g. fluorescein, rhodamine, Texas red,
etc. Final detection uses a substrate that undergoes a color change
in the presence of the peroxidase. The absence or presence of
antibody binding may be determined by various methods, including
flow cytometry of dissociated cells, microscopy, radiography,
scintillation counting, etc.
[0089] Alternatively, one may focus on the expression of DGAT.
Biochemical studies may be performed to determine whether a
sequence polymorphism in a DGAT coding region or control regions is
associated with disease. Disease associated polymorphisms may
include deletion or truncation of the gene, mutations that alter
expression level, that affect the activity of the protein, etc.
[0090] Changes in the promoter or enhancer sequence that may affect
expression levels of DGAT can be compared to expression levels of
the normal allele by various methods known in the art. Methods for
determining promoter or enhancer strength include quantitation of
the expressed natural protein; insertion of the variant control
element into a vector with a reporter gene such as
.beta.-galactosidase, luciferase, chloramphenicol
acetyltransferase, etc. that provides for convenient quantitation;
and the like.
[0091] A number of methods are available for analyzing nucleic
acids for the presence of a specific sequence, e.g. a disease
associated polymorphism. Where large amounts of DNA are available,
genomic DNA is used directly. Alternatively, the region of interest
is cloned into a suitable vector and grown in sufficient quantity
for analysis. Cells that express DGAT may be used as a source of
mRNA, which may be assayed directly or reverse transcribed into
cDNA for analysis. The nucleic acid may be amplified by
conventional techniques, such as the polymerase chain reaction
(PCR), to provide sufficient amounts for analysis. The use of the
polymerase chain reaction is described in Saiki, et al (1985),
Science 239:487, and a review of techniques may be found in
Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press
1989, pp.14.2-14.3.3. Alternatively, various methods are known in
the art that utilize oligonucleotide ligation as a means of
detecting polymorphisms, for examples see Riley et al. (1990),
Nucl. Acids Res. 18:2887-2890; and Delahunty et al. (1996), Am. J.
Hum. Genet. 58:1239-1246.
[0092] A detectable label may be included in an amplification
reaction. Suitable labels include fluorochromes, e.g. fluorescein
isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,
allophycocyanin, 6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyflu- orescein (JOE),
6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexach-
lorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive
labels, e.g. .sup.32P, .sup.35S, .sup.3H; etc. The label may be a
two stage system, where the amplified DNA is conjugated to biotin,
haptens, etc. having a high affinity binding partner, e.g. avidin,
specific antibodies, etc., where the binding partner is conjugated
to a detectable label. The label may be conjugated to one or both
of the primers. Alternatively, the pool of nucleotides used in the
amplification is labeled, so as to incorporate the label into the
amplification product.
[0093] The sample nucleic acid, e.g. amplified or cloned fragment,
is analyzed by one of a number of methods known in the art. The
nucleic acid may be sequenced by dideoxy or other methods, and the
sequence of bases compared to a wild-type DGAT sequence.
Hybridization with the variant sequence may also be used to
determine its presence, by Southern blots, dot blots, etc. The
hybridization pattern of a control and variant sequence to an array
of oligonucleotide probes immobilized on a solid support, as
described in U.S. Pat. No. 5,445,934, or in WO 95/35505, may also
be used as a means of detecting the presence of variant sequences.
Single strand conformational polymorphism (SSCP) analysis,
denaturing gradient gel electrophoresis (DGGE), and heteroduplex
analysis in gel matrices are used to detect conformational changes
created by DNA sequence variation as alterations in electrophoretic
mobility. Alternatively, where a polymorphism creates or destroys a
recognition site for a restriction endonuclease, the sample is
digested with that endonuclease, and the products size fractionated
to determine whether the fragment was digested. Fractionation is
performed by gel or capillary electrophoresis, particularly
acrylamide or agarose gels.
[0094] Screening for mutations in DGAT may be based on the
functional or antigenic characteristics of the protein. Protein
truncation assays are useful in detecting deletions that may affect
the biological activity of the protein. Various immunoassays
designed to detect polymorphisms in DGAT proteins may be used in
screening. Where many diverse genetic mutations lead to a
particular disease phenotype, functional protein assays have proven
to be effective screening tools. The activity of the encoded DGAT
protein may be determined by comparison with the wild-type
protein.
[0095] Diagnostic methods of the subject invention in which the
level of DGAT expression is of interest will typically involve
comparison of the DGAT nucleic acid abundance of a sample of
interest with that of a control value to determine any relative
differences, where the difference may be measured qualitatively
and/or quantitatively, which differences are then related to the
presence or absence of an abnormal DGAT expression pattern. A
variety of different methods for determining the nucleic acid
abundance in a sample are known to those of skill in the art, where
particular methods of interest include those described in: Pietu et
al., Genome Res. (June 1996) 6: 492-503; Zhao et al., Gene (Apr.
24, 1995) 156: 207-213; Soares, Curr. Opin. Biotechnol. (October
1997) .delta.: 542-546; Raval, J. Pharmacol Toxicol Methods
(November 1994) 32: 125-127; Chalifour et al., Anal. Biochem (Feb.
1, 1994) 216: 299-304; Stolz & Tuan, Mol. Biotechnol. (December
19960 6: 225-230; Hong et al., Bioscience Reports (1982) 2: 907;
and McGraw, Anal. Biochem. (1984) 143: 298. Also of interest are
the methods disclosed in WO 97/27317, the disclosure of which is
herein incorporated by reference.
[0096] The subject diagnostic or screening methods may be used to
identify the presence of, or predisposition to, disease conditions
associated with acylglycerol metabolism, particularly those
associated with DGAT and/or DGAT-2 activity. Such disease
conditions include: hyperlipidemia (including excess serum
triglyceride levels), cardiovascular disease, obesity, diabetes,
cancer, neurological disorders, immunological disorders, and the
like.
[0097] Also provided are methods of regulating, including enhancing
and inhibiting, DGAT activity in a host. A variety of situations
arise where modulation of DGAT activity in a host is desired, where
such conditions include disease conditions associated with DGAT
activity and non-disease conditions in which a modulation of DGAT
activity is desired for a variety of different reasons, e.g.
cosmetic weight control.
[0098] For the modulation of DGAT activity in a host, an effective
amount of active agent that modulates the activity, e.g. reduces
the activity, of DGAT in vivo, is administered to the host. The
active agent may be a variety of different compounds, including:
the polynucleotide compositions of the subject invention, the
polypeptide compositions of the subject invention, a naturally
occurring or synthetic small molecule compound, an antibody,
fragment or derivative thereof, an antisense composition, and the
like.
[0099] The nucleic acid compositions of the subject invention find
use as therapeutic agents in situations where one wishes to enhance
DGAT activity in a host, e.g. in a mammalian host in which DGAT
activity is low resulting in a disease condition, etc. The DGAT
genes, gene fragments, or the encoded DGAT protein or protein
fragments are useful in gene therapy to treat disorders associated
with DGAT defects. Expression vectors may be used to introduce the
DGAT gene into a cell. Such vectors generally have convenient
restriction sites located near the promoter sequence to provide for
the insertion of nucleic acid sequences. Transcription cassettes
may be prepared comprising a transcription initiation region, the
target gene or fragment thereof, and a transcriptional termination
region. The transcription cassettes may be introduced into a
variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus;
adenovirus; and the like, where the vectors are able to transiently
or stably be maintained in the cells, usually for a period of at
least about one day, more usually for a period of at least about
several days to several weeks.
[0100] Naturally occurring or synthetic small molecule compounds of
interest include numerous chemical classes, though typically they
are organic molecules, preferably small organic compounds having a
molecular weight of more than 50 and less than about 2,500 daltons.
Candidate agents comprise functional groups necessary for
structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Of particular interest are those agents
identified by the screening assays of the subject invention, as
described above.
[0101] Also of interest as active agents are antibodies that
modulate, e.g. reduce, if not inhibit, DGAT activity in the host.
Suitable antibodies are obtained by immunizing a host animal with
peptides comprising all or a portion of a DGAT protein, such as the
DGAT polypeptide compositions of the subject invention. Suitable
host animals include mouse, rat sheep, goat, hamster, rabbit, etc.
The origin of the protein immunogen may be mouse, human, rat,
monkey etc. The host animal will generally be a different species
than the immunogen, e.g. human DGAT used to immunize mice, etc.
[0102] The immunogen may comprise the complete protein, or
fragments and derivatives thereof. Preferred immunogens comprise
all or a part of DGAT, where these residues contain the
post-translation modifications, such as glycosylation, found on the
native DGAT. Immunogens comprising the extracellular domain are
produced in a variety of ways known in the art, e.g. expression of
cloned genes using conventional recombinant methods, isolation from
HEC, etc.
[0103] For preparation of polyclonal antibodies, the first step is
immunization of the host animal with DGAT, where the DGAT will
preferably be in substantially pure form, comprising less than
about 1% contaminant. The immunogen may comprise complete DGAT,
fragments or derivatives thereof. To increase the immune response
of the host animal, the DGAT may be combined with an adjuvant,
where suitable adjuvants include alum, dextran, sulfate, large
polymeric anions, oil & water emulsions, e.g. Freund's
adjuvant, Freund's complete adjuvant, and the like. The DGAT may
also be conjugated to synthetic carrier proteins or synthetic
antigens. A variety of hosts may be immunized to produce the
polyclonal antibodies. Such hosts include rabbits, guinea pigs,
rodents, e.g. mice, rats, sheep, goats, and the like. The DGAT is
administered to the host, usually intradermally, with an initial
dosage followed by one or more, usually at least two, additional
booster dosages. Following immunization, the blood from the host
will be collected, followed by separation of the serum from the
blood cells. The Ig present in the resultant antiserum may be
further fractionated using known methods, such as ammonium salt
fractionation, DEAE chromatography, and the like.
[0104] Monoclonal antibodies are produced by conventional
techniques. Generally, the spleen and/or lymph nodes of an
immunized host animal provide a source of plasma cells. The plasma
cells are immortalized by fusion with myeloma cells to produce
hybridoma cells. Culture supernatant from individual hybridomas is
screened using standard techniques to identify those producing
antibodies with the desired specificity. Suitable animals for
production of monoclonal antibodies to the human protein include
mouse, rat, hamster, etc. To raise antibodies against the mouse
protein, the animal will generally be a hamster, guinea pig,
rabbit, etc. The antibody may be purified from the hybridoma cell
supernatants or ascites fluid by conventional techniques, e.g.
affinity chromatography using DGAT bound to an insoluble support,
protein A sepharose, etc.
[0105] The antibody may be produced as a single chain, instead of
the normal multimeric structure. Single chain antibodies are
described in Jost et al. (1994) J.B.C. 269:26267-73, and others.
DNA sequences encoding the variable region of the heavy chain and
the variable region of the light chain are ligated to a spacer
encoding at least about 4 amino acids of small neutral amino acids,
including glycine and/or serine. The protein encoded by this fusion
allows assembly of a functional variable region that retains the
specificity and affinity of the original antibody.
[0106] For in vivo use, particularly for injection into humans, it
is desirable to decrease the antigenicity of the antibody. An
immune response of a recipient against the blocking agent will
potentially decrease the period of time that the therapy is
effective. Methods of humanizing antibodies are known in the art.
The humanized antibody may be the product of an animal having
transgenic human immunoglobulin constant region genes (see for
example International Patent Applications WO 90/10077 and WO
90/04036). Alternatively, the antibody of interest may be
engineered by recombinant DNA techniques to substitute the CH1,
CH2, CH3, hinge domains, and/or the framework domain with the
corresponding human sequence (see WO 92/02190).
[0107] The use of Ig cDNA for construction of chimeric
immunoglobulin genes is known in the art (Liu et al. (1987)
P.N.A.S. 84:3439 and (1987) J. Immunol. 139:3521). mRNA is isolated
from a hybridoma or other cell producing the antibody and used to
produce cDNA. The cDNA of interest may be amplified by the
polymerase chain reaction using specific primers (U.S. Pat. Nos.
4,683,195 and 4,683,202). Alternatively, a library is made and
screened to isolate the sequence of interest. The DNA sequence
encoding the variable region of the antibody is then fused to human
constant region sequences. The sequences of human constant regions
genes may be found in Kabat et al. (1991) Sequences of Proteins of
Immunological Interest, N.I.H. publication no. 91-3242. Human C
region genes are readily available from known clones. The choice of
isotype will be guided by the desired effector functions, such as
complement fixation, or activity in antibody-dependent cellular
cytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of
the human light chain constant regions, kappa or lambda, may be
used. The chimeric, humanized antibody is then expressed by
conventional methods.
[0108] Antibody fragments, such as Fv, F(ab').sub.2 and Fab may be
prepared by cleavage of the intact protein, e.g. by protease or
chemical cleavage. Alternatively, a truncated gene is designed. For
example, a chimeric gene encoding a portion of the F(ab').sub.2
fragment would include DNA sequences encoding the CH1 domain and
hinge region of the H chain, followed by a translational stop codon
to yield the truncated molecule.
[0109] Consensus sequences of H and L J regions may be used to
design oligonucleotides for use as primers to introduce useful
restriction sites into the J region for subsequent linkage of V
region segments to human C region segments. C region cDNA can be
modified by site directed mutagenesis to place a restriction site
at the analogous position in the human sequence.
[0110] Expression vectors include plasmids, retroviruses, YACs, EBV
derived episomes, and the like. A convenient vector is one that
encodes a functionally complete human CH or CL immunoglobulin
sequence, with appropriate restriction sites engineered so that any
VH or VL sequence can be easily inserted and expressed. In such
vectors, splicing usually occurs between the splice donor site in
the inserted J region and the splice acceptor site preceding the
human C region, and also at the splice regions that occur within
the human CH exons. Polyadenylation and transcription termination
occur at native chromosomal sites downstream of the coding regions.
The resulting chimeric antibody may be joined to any strong
promoter, including retroviral LTRs, e.g. SV-40 early promoter,
(Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus
LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine
leukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native
Ig promoters, etc.
[0111] In yet other embodiments of the invention, the active agent
is an agent that modulates, and generally decreases or down
regulates, the expression of DGAT in the host. Antisense molecules
can be used to down-regulate expression of DGAT in cells. The
anti-sense reagent may be antisense oligonucleotides (ODN),
particularly synthetic ODN having chemical modifications from
native nucleic acids, or nucleic acid constructs that express such
anti-sense molecules as RNA. The antisense sequence is
complementary to the mRNA of the targeted gene, and inhibits
expression of the targeted gene products. Antisense molecules
inhibit gene expression through various mechanisms, e.g. by
reducing the amount of mRNA available for translation, through
activation of RNAse H, or steric hindrance. One or a combination of
antisense molecules may be administered, where a combination may
comprise multiple different sequences.
[0112] Antisense molecules may be produced by expression of all or
a part of the target gene sequence in an appropriate vector, where
the transcriptional initiation is oriented such that an antisense
strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a synthetic oligonucleotide. Antisense oligonucleotides
will generally be at least about 7, usually at least about 12, more
usually at least about 20 nucleotides in length, and not more than
about 500, usually not more than about 50, more usually not more
than about 35 nucleotides in length, where the length is governed
by efficiency of inhibition, specificity, including absence of
cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and
selective inhibitors of gene expression (see Wagner et al. (1996),
Nature Biotechnol. 14:840-844).
[0113] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in an in
vitro or animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0114] Antisense oligonucleotides may be chemically synthesized by
methods known in the art (see Wagner et al. (1993), supra, and
Milligan et al., supra.) Preferred oligonucleotides are chemically
modified from the native phosphodiester structure, in order to
increase their intracellular stability and binding affinity. A
number of such modifications have been described in the literature,
which alter the chemistry of the backbone, sugars or heterocyclic
bases.
[0115] Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.alpha.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .beta.-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
[0116] As an alternative to anti-sense inhibitors, catalytic
nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc.
may be used to inhibit gene expression. Ribozymes may be
synthesized in vitro and administered to the patient, or may be
encoded on an expression vector, from which the ribozyme is
synthesized in the targeted cell (for example, see International
patent application WO 9523225, and Beigelman et al. (1995), Nucl.
Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic
activity-are described in WO 9506764. Conjugates of anti-sense ODN
with a metal complex, e.g. terpyridylCu(II), capable of mediating
mRNA hydrolysis are described in Bashkin et al. (1995), Appl.
Biochem. Biotechnol. 54:43-56.
[0117] As mentioned above, an effective amount of the active agent
is administered to the host, where "effective amount" means a
dosage sufficient to produce a desired result, where the desired
result in the desired modulation, e.g. enhancement, reduction, of
DGAT activity.
[0118] In the subject methods, the active agent(s) may be
administered to the host using any convenient means capable of
resulting in the desired effect. Thus, the agent can be
incorporated into a variety of formulations for therapeutic
administration. More particularly, the agents of the present
invention can be formulated into pharmaceutical compositions by
combination with appropriate, pharmaceutically acceptable carriers
or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants and aerosols.
[0119] As such, administration of the agents can be achieved in
various ways, including oral, buccal, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.,
administration.
[0120] In pharmaceutical dosage forms, the agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0121] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0122] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0123] The agents can be utilized in aerosol formulations to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0124] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0125] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0126] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0127] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0128] Where the agent is a polypeptide, polynucleotide, analog or
mimetic thereof, e.g. antisense composition, it may be introduced
into tissues or host cells by any number of routes, including viral
infection, microinjection, or fusion of vesicles. Jet injection may
also be used for intramuscular administration, as described by
Furth et al. (1992), Anal Biochem 205:365-368. The DNA may be
coated onto gold microparticles, and delivered intradermally by a
particle bombardment device, or "gene gun" as described in the
literature (see, for example, Tang et al. (1992), Nature
356:152-154), where gold microprojectiles are coated with the DGAT
DNA, then bombarded into skin cells.
[0129] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means.
[0130] The subject methods find use in the treatment of a variety
of different disease conditions involving acylglycerol metabolism,
and particularly DGAT activity, including both insufficient or
hypo-DGAT activity and hyper-DGAT activity. Representative diseases
that may be treated according to the subject methods include:
hyperlipidemia (including excess serum triglyceride levels),
cardiovascular disease, obesity, diabetes, cancer, neurological
disorders, immunological disorders, skin disorders associated with
sebaceous gland activity, e.g. acne, and the like.
[0131] By treatment is meant at least an amelioration of the
symptoms associated with the pathological condition afflicting the
host, where amelioration is used in a broad sense to refer to at
least a reduction in the magnitude of a parameter, e.g. symptom,
associated with the pathological condition being treated, such as
serum triglyceride level, weight, total body fat content, etc. As
such, treatment also includes situations where the pathological
condition, or at least symptoms associated therewith, are
completely inhibited, e.g. prevented from happening, or stopped,
e.g. terminated, such that the host no longer suffers from the
pathological condition, or at least the symptoms that characterize
the pathological condition. For example, where the disease
condition is marked by the presence of elevated lipid levels,
treatment includes at least a reduction in the observed lipid
levels, including a restoration of normal lipid levels. As another
example, where the disease is obesity, treatment results in at
least a reduction in the overall weight and/or total body fat
content of the host.
[0132] The subject methods also find use in the modulation of DGAT
activity in hosts not suffering from a disease condition but in
which the modulation of DGAT activity is nonetheless desired. For
example, sperm production in males has been associated with DGAT
activity. As such, in males where at least reduced sperm production
is desired, the subject methods can be used to reduce DGAT activity
in such males, e.g. by administering an agent that reduces DGAT
activity in such males, where such agents are described above. In
other words, the subject methods provide a means of male
contraception. Alternatively, where increased sperm count in a
given male is desired, e.g. in those conditions where the male has
reduced fertility, the subject methods can be used to enhance DGAT
activity in the male and thereby increase sperm count and
fertility, e.g. by administering to the male host a DGAT enhancing
agent, as described above.
[0133] A variety of hosts are treatable according to the subject
methods. Generally such hosts are "mammals" or "mammalian," where
these terms are used broadly to describe organisms which are within
the class mammalia, including the orders carnivore (e.g., dogs and
cats), rodentia (e.g., mice, guinea pigs, and rats), and primates
(e.g., humans, chimpanzees, and monkeys). In many embodiments, the
hosts will be humans.
[0134] Kits with unit doses of the active agent, usually in oral or
injectable doses, are provided. In such kits, in addition to the
containers containing the unit doses will be an informational
package insert describing the use and attendant benefits of the
drugs in treating pathological condition of interest. Preferred
compounds and unit doses are those described herein above.
[0135] Methods and Compositions for Producing Triglycerides and
Triglyceride Compositions
[0136] Also provided by the subject invention are methods for
preparing triglycerides and triglyceride comprising compositions,
as well as the compositions produced by these methods. In preparing
triglycerides with the subject invention, at least the direct
substrates of the desired triacylglyercol, e.g. diacylglycerol and
fatty acyl CoA, will be combined in the presence of the polypeptide
under conditions sufficient for the acylation of the diacylglycerol
to occur. The synthesis may occur in an in vitro system, e.g. in a
vessel in which the substrates or precursors thereof and the DGAT
enzyme, as well as any other requisite enzymes (e.g. as need to
convert the substrate precursors to substrates), or an in vivo
system, e.g. a host cell that naturally comprises the substrates
and into which a DGAT gene has been inserted in a manner sufficient
for expression of the DGAT gene, where the resultant triglyceride
products may be separated from the host cell using standard
separation techniques.
[0137] Of interest for use in producing triglyceride compositions
are transgenic plants/fungi that have been genetically manipulated
using the nucleic acid compositions of the subject invention to
produce triglycerides and/or compositions thereof in one or more
desirable ways. Transgenic plants/fungi of the subject invention
are those plants/fungi that at least: (a) produce more triglyceride
or triglyceride composition than wild type, e.g. produce more oil,
such as by producing seeds having a higher oil content, as compared
to wild-type; (b) produce triglyceride compositions, e.g. oils,
that are enriched for triglycerides and/or enriched for one or more
particular triglycerides as compared to wild type; and the like. Of
interest are transgenic plants that produce commercially valuable
triglyceride compositions or oils, such as canola, rapeseed, palm,
corn, etc., containing various poly- and mono-unsaturated fatty
acids, and the like. Of particular interest are transgenic plants,
such as canola, rapeseed, palm, oil, etc., which have been
genetically modified to produce seeds having higher oil content
than the content found in the corresponding wild type, where the
oil content of the seeds produced by such plants is at least 10%
higher, usually at least 20% higher, and in many embodiments at
least 30% higher than that found in the wild type, where in many
embodiments seeds having oil contents that are 50% higher, or even
greater, as compared to seeds produced by the corresponding
wild-type plant, are produced. The seeds produced by such DGAT
transgenic plants can be used as sources of oil or as sources of
additional DGAT transgenic plants. Such transgenic plants and seeds
therefore find use in methods of producing oils. In such methods,
DGAT transgenic plants engineered to produce seeds having a higher
oil content than the corresponding wild-type, e.g. seeds in which
the DGAT gene is overexpressed, are grown, the seeds are harvested
and then processed to recover the oil. The subject transgenic
plants can also be used to produce novel oils characterized by the
presence of triglycerides in different amounts and/or ratios than
those observed in naturally occurring oils. The transgenic
plants/fungi described above can be readily produced by those of
skill in the art armed with the nucleic acid compositions of the
subject invention. See the discussion on how to prepare transgenic
plants, supra.
[0138] The triglyceride compositions described above find use in a
variety of different applications. For example, such compositions
or oils find use as food stuffs, being used as ingredients,
spreads, cooking materials, etc. Alternatively, such oils find use
as industrial feedstocks for use in the production of chemicals,
lubricants, surfactants and the like.
[0139] Also of interest are transgenic non-human animals suitable
for use as sources of food products and/or animal based industrial
products. Such trans-genic non-human animals, e.g. transgenic mice,
rats, livestock, such as cows, pigs, horses, birds, etc, may be
produced using methods known in the art and reviewed supra. Such
trans-genic non-human animals can be used for sources of a variety
of different food and industrial products in which the triglyceride
content is specifically tailored in a desirable manner. For
example, such trans-genic animals that have been modified in a
manner such that DGAT activity is reduced as compared to the wild
type can be used as sources of food products that are low in
triglyceride content, e.g. low fat or lean meat products, low fat
milk, low fat eggs, and the like.
[0140] The following examples are offered primarily for purposes of
illustration. It will be readily apparent to those skilled in the
art that the formulations, dosages, methods of administration, and
other parameters of this invention may be further modified or
substituted in various ways without departing from the spirit and
scope of the invention.
EXPERIMENTAL
[0141] I. Identification and Characterization of DGAT
[0142] A. Cloning of DGAT cDNA.
[0143] ESTs (accession numbers R07932 (human) and W10786 (mouse))
with sequence similarity to ACAT were identified from BLAST
searches of the databases. The 5' end of the DGAT cDNA was obtained
by using 5' RACE and a mouse spleen Marathon Ready.TM. cDNA library
(Clontech, Palo Alto, Calif.). The sequences were deposited in
GenBank (accession no. AF078752).
[0144] The translation of a full-length cDNA for the mouse EST (SEQ
ID NO.: 02) predicts an open reading frame encoding a 498-amino
acid protein that is 20% identical to mouse ACAT, with the most
highly conserved regions in the C-terminus.
[0145] The predicted protein sequence (SEQ ID NO:05) contains a
potential N-linked glycosylation site and a putative tyrosine
phosphorylation site. A serine residue found in ACAT that is
necessary for enzyme activity (as reported in Cao et al., J. Biol.
Chem. (1996) 271:14642-14648) appears to be conserved. The protein
has multiple hydrophobic domains and 6-12 possible transmembrane
domains. Analysis by a transmembrane region prediction program
(http://ulrec3.unil.ch/software/TMPRED_form.html) favors nine
transmembrane domains (amino acids 96-114, 140-157, 174-198,
200-218, 293-311, 337-360, 412-434, 436-456, and 461-484).
[0146] B. Insect cell expression studies. DGAT coding sequences
with or without an N-terminal FLAG epitope (IBI, Kodak, New Haven,
Conn.) (MGDYKDDDDG-, epitope underlined (SEQ ID NO:08)) were
subcloned into the baculovirus transfer vector pVL1392 (PharMingen,
San Diego, Calif.). High titers of recombinant baculoviruses were
obtained by cotransfection of ACAT or DGAT baculovirus transfer
vectors with viral BaculoGold.TM. DNA (PharMingen), followed by
plaque purification and virus amplification in Sf9 cells (cultured
in Grace's medium (Life Technologies, Grand Island, N.Y.) and 10%
fetal bovine serum). H5 insect cells (cultured in serum-free
Express-Five medium (Life Technologies)) were plated on day 0
(8.5.times.10.sup.6 cells/100-mm dish) and infected on day 1 with
high titers of virus at a multiplicity of infection (MOI) that was
empirically determined. On day 3, cells were collected by
centrifugation and washed twice with phosphate-buffered saline
(PBS). Cell pellets were homogenized by 10 passages through a 27-G
needle in 0.1 M sucrose, 50 mM Kcl, 40 mM KH.sub.2PO.sub.4, 30 mM
EDTA (pH 7.2). Total membrane fractions (100,000.times.g pellet)
were resuspended in the homogenization buffer and frozen
(-80.degree. C.). Immunoblots of membrane proteins (75 .mu.g) were
performed using the anti-FLAG M2 monoclonal antibody (IBI,
Kodak).
[0147] For metabolic labeling experiments, H5 insect cells were
plated on day 0 at 2.9.times.10.sup.6 cells per 60-mm dish and
infected on day 1 with high titers of viruses. On day 3, cells were
washed and incubated in methionine- and cysteine-free medium (SF900
II, Life Technologies) for 2 h, followed by incubation in the same
medium containing 715 .mu.Ci of [.sup.35S]methionine and
[.sup.35S]cysteine (Amersham Pro-Mix) for 1 h. Cells were washed
twice with PBS, collected by low-speed centrifugation, and the cell
pellet was resuspended in 0.5 ml of 50 mM Tris-HCl, 150 mM NaCl, 5
mM EDTA, 1 mM PMSF, 1% Triton X-100 (pH 7.4) and sonicated.
Cellular protein samples (100 mg) were analyzed by SDS-PAGE and
autoradiography.
[0148] For ACAT assays, cell-membrane proteins (100 .mu.g) were
assayed by using [1-.sup.14C]oleoyl CoA (51 mCi/mmol, Amersham,
Arlington Heights, Ill.) and cholesterol/egg phosphatidyl choline
(PC) liposomes (molar ratio=0.7) as described in Smith et al., J.
Lipids Res. (1995) 36:641-652. In some assays, other acyl acceptors
were substituted for cholesterol in the liposomes at a 0.2 molar
ratio (acceptor:egg PC) to test their ability to act as substrates.
Incorporation of the [.sup.14C]oleoyl group into products was
assessed by thin-layer chromatography, followed by autoradiography.
DGAT assays were based on assays optimized for rat liver. See
Andersson supra, Ozasa et al., J. Lipids Res. (1989) 30:1759-1762.
The incorporation of [.sup.14C]oleoyl CoA into triacylglycerol was
measured under apparent V.sub.MAX conditions by using exogenous
diacylglycerol provided as diacylglycerol:egg PC liposomes (molar
ratio 0.16). Cell-membrane proteins (20-25 .mu.g) were assayed in
0.25 M sucrose, 1 mM EDTA, 150 mM MgCl.sub.2, 100 mM Tris-HCl (pH
7.5) containing 250 .mu.g of bovine serum albumin and 20 .mu.g of
diacylglycerol in liposomes (final volume=0.2 ml) and 5 mmol
[.sup.14C]oleoyl CoA (40,000 dpm/nmol). Reactions were carried out
for 5 min, and the products analyzed as described in Erickson et
al., J. Lipids Res. (1980) 21: 930-941. Similar assays were
performed with 1-stearoyl-2-[1-.sup.14C]arachidonyl-sn-glycerol (53
mCi/mmol, Amersham) diluted to a final activity of 38,000 dpm/nmol
with unlabeled 1,2-diacyl-sn-glycerol and unlabeled oleoyl CoA.
[0149] Relative triacylglycerol and DAG masses were determined by
total lipid extraction of membranes or cells followed by thin-layer
chromatography, iodine vapor visualization, photography of the
plates, and densitometric analysis. Triolein standards were used to
estimate mass of triacylglycerols, and DAG units were estimated
relative to one another. Triacylglycerol values were normalized to
1 for wild-type virus-infected cell membranes to correct for
interexperiment variability.
[0150] Cells infected with the virus containing DGAT cDNA expressed
a .about.47-kDa protein at high levels in the membrane fraction,
but lacked detectable cholesterol esterification activity as
compared with ACAT virus-infected cells. Using a variety of other
possible acyl acceptors as provided substrates (including
25-hydroxy-, 27-hydroxy-, 7.alpha.-hydroxy- or
7.beta.-hydroxycholesterols, 7-ketocholesterol, vitamins D2 and D3,
vitamin E, dehydroepiandrosterone, retinol, ethanol, sitosterol,
lanosterol, and ergosterol), no acyltransferase activity was
detected in H5 membranes expressing the protein, as assessed by
autoradiography of thin-layer chromatography plates used to analyze
reaction products. However, further analysis of these plates
revealed that membranes from these cells had significantly
increased triacylglycerol mass (as assessed by 12 visualization)
and incorporated significantly more [.sup.14C]oleoyl CoA into
triacylglycerols than did membranes from wild-type virus-infected
cells (197 vs. 55 pmol/mg prot/min). These data suggested that the
identified cDNA might encode a DGAT.
[0151] Measurements of DGAT activity in membranes from H5 insect
cells expressing the putative DGAT cDNA revealed that DGAT activity
in these membranes was more than fivefold higher than that in
membranes from wild-type virus-infected cells. The DGAT activity
level increased proportionately with the amount of FLAG-tagged
protein expressed in membranes isolated from cells harvested at
different time points following infection. DGAT activity levels
were similar regardless of whether [.sup.14C]diacylglycerol or
[.sup.14C]oleoyl CoA was used as the labeled substrate. In the
absence of added oleoyl CoA, [.sup.14C]diacylglycerol was not
incorporated into triacylglycerols. Additionally, [.sup.3H]oleic
acid was not incorporated into triacylglycerols in DGAT
virus-infected membranes (7.+-.6 vs. 49.+-.47 pmol
triacylglycerol/mg prot/min for wild-type, n=3), establishing the
requirement for a fatty acyl CoA. Triacylglycerol mass was more
than 10-fold higher in membranes from DGAT virus-infected cells
than in membranes from wild-type virus-infected cells (11.+-.7 vs.
1.+-.0.5 pg/.mu.g membrane protein, P=0.04, n=5). No change in
relative DAG mass was observed (0.33.+-.0.05 vs. 0.34.+-.0.12 units
for DGAT and wild-type, respectively).
[0152] C. mRNA expression. Human Multiple Tissue northern blots
(Clontech) were hybridized with a .sup.32P-labeled 1.1-kb human
DGAT fragment from the human EST. For mouse tissues, total RNA was
prepared with Trizol reagent (Life Technologies), and samples (10
mg) were analyzed by northern blotting using a .sup.32P-labeled,
1-kb mouse DGAT fragment from the mouse EST. Blots were stripped
and sequentially reprobed for G3PDH and 28S RNA as described in
Barbu & Dautry, Nucleic Acids Res. (1989)17:7115. Bands in
autoradiograms from the 3T3-L1 experiments were quantified with a
phosphoimager (Fuji Medical Systems, Stamford, Conn.).
[0153] mRNA expression was detected in every human and mouse tissue
examined, as expected for DGAT's role in cellular glycerolipid
metabolism. The highest expression levels were found in the small
intestine, consistent with a proposed role for DGAT in intestinal
fat absorption (see Brindley supra, and Mansbach, Biochim. Biophys.
Acta (1973) 296:386-402. Additionally, mRNA was expressed in mouse
adipose tissue, a tissue known to have high DGAT activity (see
Coleman & Bell, supra). Interestingly, mRNA expression was not
particularly high in the livers of humans or mice, despite the fact
that liver tissue possesses DGAT activity (as reported in Polokoff
& Bell, Biochim. Biophys. Acta (1980)35:535-545.
[0154] D. NIH 3T3-L1 differentiation. NIH 3T3-L1 fibroblasts were
cultured in Dulbecco's Modified Eagle's medium supplemented with
10% fetal bovine serum, 100 units/ml penicillin, 100 .mu.g/ml
streptomycin, and 2 mM L-glutamine. The differentiation of 3T3-L 1
cells into adipocytes was induced by incubating confluent
monolayers of cells in serum-containing medium supplemented with
10-5 M dexamethasone, 0.5 mM isobutylmethylxanthine, and 10
.mu.g/ml insulin as described Brasaemle et al., J. Lipid Res.
(1997) 38:2249-2263.
[0155] It was found that mRNA expression increased markedly in
parallel with DGAT activity in NIH 3T3-L1 cells during their
differentiation into adipocytes, a process known to be associated
with increases in DGAT activity (see Coleman et al., J. Biol. Chem.
(1978) 253:7256-7261) and triacylglycerol mass accumulation (see
Green & Kehinde, Cell (1975) 5:19-27.
[0156] E. Intermediate Conclusions
[0157] The above results indicate that the cDNA encodes a DGAT
protein. As a final piece of evidence confirming the identity of
this cDNA, the mouse DGAT gene in embryonic stem cells was
disrupted and germline transmission of this mutation was acheived.
In preliminary experiments, the DGAT activity in membranes from
embryonic fibroblasts homozygous for the knockout mutation (-/-)
are markedly reduced compared with that in wild-type fibroblasts
(25.5.+-.2.6 vs. 453.6.+-.4.5 pmol triacylglycerol/mg prot/min for
-/- and +/+, respectively; P<0.001). Taken together, the
experimental data indicate that the identified cDNA encodes a DGAT
catalytic unit.
[0158] F. Gene mapping. Primers derived from the human EST
sequences were used to identify genomic clones in an arrayed BAC
library according to manufacturer's protocol (Research Genetics,
Huntsville, Ala.). The BAC clone was mapped to chromosome 8qter by
fluorescent in situ hybridization as described in Stokke et al.,
Genomics (1995) 26:134-137. The clone (RMC08P049) may be requested
from the website (http://rmc-www.lbl.gov). Linkage analysis for
mouse gene mapping was performed with a panel of 67 progeny derived
from a ((C57BL/6J.times.Mus spretus) F1.times.C57BL/6J)
interspecific backcross as described in Warden et al., Genomics
(1993) 18:295-307. This backcross panel has been typed for more
than 400 loci distributed throughout the genome. See Welch et al.,
J. Lipid Res. (1996) 37:1406-1421. Briefly, parental strain DNAs
were screened for restriction fragment-length variants by
restriction enzyme digestion and hybridization with a radiolabeled,
1-kb mouse DGAT cDNA fragment as described in Warden, supra.
Filters were washed in 1.0.times.SSC/0.1% SDS, at 50.degree. C.,
for 20 min. Autoradiograms were exposed for 3 days at -70.degree.
C. Linkage to previously typed chromosomal markers was detected by
using Map Manager v.2.6.5, and loci were ordered by minimizing the
number of recombination events between DGAT and the markers. See
Manly, Mamm. Genome (1993)4:303-313. The mouse homolog for the DGAT
gene (DGAT) was mapped to a region of chromosome 15 that exhibits
homology with human chromosome 8. The mouse DGAT gene was found to
be colocalized with quantitative trait loci for plasma levels of
triacylglycerol-rich lipoproteins.
[0159] II. Identification of DGAT cDNA from Arabidopsis thaliana.
The plant (A. thaliana) DGAT gene (#AA042298) (SEQ ID NO:03) was
identified from BLAST searches of the EST database using mouse DGAT
sequences as a probe. The plant DGAT EST protein sequences encoded
by plant DGAT genes are 40-50% identical to mammalian DGAT enzymes.
Furthermore, the plant DGAT sequences are more closely related to
other mammalian DGAT sequences than to ACAT protein sequences.
[0160] III. Transgenic Animal Studies
[0161] A. Preparation and Characterization of DGAT Knockout
Mice.
[0162] DGAT knockout mice were generated using standard techniques
of gene targeting. A mouse P1 clone containing the mouse DGAT gene
was isolated from a genomic 129/Sv library. Short and long arms of
homologous sequences were amplified by PCR from this clone and
subcloned intopNTKLoxP to generate a gene targeting vector. The
vector contained a neomycin resistance gene for positive selection
and a thymidine kinase gene for negative selection. Upon homologous
recombination, the vector was designed to interrupt the DGAT coding
sequences at amino acid 360 of the 498-amino acid murine protein.
The entire C-terminus, including a highly conserved region common
to all ACAT gene family members is deleted. The gene targeting
vector was electroporated into RF8 embryonic stem cells by
electroporation, and several targeted clones were identified by
Southern blotting (targeting frequency of .about.1 in 300). The
above strategy is depicted in FIG. 1.
[0163] One of these targeted clones was injected into C57BL/6
blastocysts and chimeras were generated; male chimeras subsequently
passed the DGAT knockout mutation through the germline to their
offspring. The resultant mice, which were heterozygous for the DGAT
gene disruption, were intercrossed to generate mice that were
homozygotes.
[0164] Inactivation of the DGAT gene in the homozygote knockouts
was verified by examining DGAT mRNA which was found to be absent in
the knockout mice. In activation of the DGAT gene was also verified
by studying DGAT activity in tissues using an assay that measures
the incorporation of [14C]oleoyl CoA into triglycerides. The
results from the activity assays are provided in FIG. 2, and show
that DGAT activity is virtually gone from every nearly every tissue
tested.
[0165] B. Phenotypic Studies
[0166] 1. General
[0167] The resultant DGAT knockout mice were viable, ostensibly
healthy, and fertile.
[0168] 2. Fat Analysis
[0169] DGAT knockout mice were leaner than their wild type
counterparts. This appeared to be true more for males than for
females. An analysis of fat pads of male mice fed a chow diet for
several months is provided in FIG. 3. Analysis of the fat pads from
different regions shows that fat pad weights trend downward in all
cases except for the mesenteric fat in females. These data indicate
that although chow fed knockout mice weighed the same as chow fed
wild type mice, See FIG. 4, the knockout mice are leaner and have
reduced adipose tissue. The reproductive and retroperitoneal fat
pad sizes are clearly reduced in size (by 50% or more) as compared
with those from wild type male mice (FIG. 3). The reduction in fat
pads in females appears be not as prominent. Mesenteric fat
(visceral fat located in the abdominal cavity) is also clearly
reduced in male mice. This is of interest as high levels of
mesenteric fat in humans have been associated with insulin
resistance, and related syndromes that carry increased risk of
heart disease. As such, inhibitors of DGAT activity are useful in
the treatment of disease conditions such as insulin resistance and
related syndromes, such as those that increased risk of heart
disease.
[0170] When the knockout mice were fed a diet rich in fat (42% of
calories as fat), they were protected from developing dietary
induced obesity, as shown in FIG. 5. Wherease wild-type mice
increased their weight to 45-50 grams on this diet, the knockout
mice were protected from developing obesity and, in fact,
maintained their body weight in the range of chow fed animals. This
protection from dietary induced obesity occurs both in male and
female knockout mice. This reduction in body weight, as compared
with wild type mice, is primarily due to decreases in adipose
tissue, as shown in FIGS. 6A and 6B. FIG. 6A shows that body weight
is significantly decreased in DGAT knockout female mice fed a
high-fat diet. In addition, body mass index is significantly
decreased. FIG. 6B illustrates that the decrease in weight occurs
primarily from a loss of fat-pad mass, although there is a small
but significant loss of liver mass as well. Thus, the protection
from dietary induced obesity in the DGAT knockout mice results from
lower amounts of adipose tissue. As such, inhibitors of DGAT
activity are useful in the treatment of dietary induced obesity and
disease conditions associated therewith.
[0171] Histologic sections of adipose tissue from both wild-type
and knockout mice fed a chow diet were also examined and compared.
The adipocytes in the fat pads of the knockout mice were reduced in
diameter as compared to those observed from the wild-type mice,
indicating that the DGAT mutation leads to smaller fat cells.
[0172] DGAT knockout mice were also crossed into mice carrying the
Agouti mutation, a dominant mutation in mice that leads to yellow
fur and obesity from overeating. Mice carrying the Agouti mutation
and wild type at the DGAT locus were larger than mice carrying the
Agouti mutation and -/- at the DGAT locus. These results indicated
that the DGAT knockout mice are protected from genetic forms of
obesity. As such, inhibitors of DGAT activity are useful in the
treatment of genetically induced obesity.
[0173] The livers of both wild type and DGAT knockout mice fed a
high fat diet were also compared. While the wild-type mice
developed large lipid droplets in their livers, the DGAT knockout
mice showed much less fat accumulation in the liver, indicating
that the DGAT knockout mice were protected from the development of
fatty liver.
[0174] In sum, the above results demonstrate that inhibitors of
DGAT lower fat stores and protect against diet-induced obesity. As
mice completely lacking DGAT are healthy, such inhibitors are of
low toxicity.
[0175] FIG. 7 illustrates that the DGAT knockout mutation has no
apparent influence on plasma glucose or free-fatty acid levels. In
addition, no differences were detected in insulin levels in the
knockout mice as compared to wild-type controls. These results
indicate that the DGAT knockout mutation does not affect glucose
metabolism in an adverse way. This data further indicates that
reduction or elimination of DGAT activity reduces adipose tissue
and improves glucose metabolism by causing diminished insulin
resistance. As such, inhibitors of DGAT find use in therapy to
treat insulin resistance or diabetes associated with obesity.
[0176] 3. Chylomicron Formation
[0177] It was also observed that chylomicron formation by the small
intestine is impaired in the DGAT knockout mice. Chylomicrons were
examined in the bloodstream by agarose gel electrophoresis after
administering a corn oil bolus to the mice. It was found that 3
knockout mice had significantly less chylomicrons than the wild
type mice. These results indicate that DGAT plays a role in
intestinal lipid absorption and that DGAT inhibitors may have a
role in diminishing intestinal fat absorption. In sum, the above
results indicate that DGAT inhibitors find use as therapeutic
agents to lower body fat content.
[0178] 4. Fur Patterns
[0179] 2 sets of DGAT+/+ and -/- male mice at age about 3 months
(young adult), 3-5 in each group per set, were studied. The -/-
mice were confirmed to be so by Southern blot analysis. Upon visual
examination, the -/- mice appeared normal except that the -/- mice
in the second group had some patchy fur loss on the back, mostly in
the intrascapular region, the degree of which was, however, highly
individual. The mice lacked oils created in the skin that coat the
hair, which was due to DGAT deficiency in the sebaceous glands,
which are atrophic in the knockout mice. As such, DGAT inhibitors
can be used to inhibit skin oil production and are therefore useful
in the treatment of acne.
[0180] 5. Wound Healing
[0181] Older females (.about.8 months old) 2 animals in each group
(+/+ and -/-) were examined. One animal had been severely bitten so
sacrifice was delayed until the mouse healed (injury results in
changes in triglyceride metabolism). She appeared to heal normally
suggesting that the DGAT gene that was inactivated in the -/- mice
was not essential to the healing process.
[0182] 6. Lactation
[0183] The other female gave birth to a healthy appearing litter.
However, the pups died soon after birth because they could not
suckle (as shown by lack of milk in the pups' stomachs/intestines).
On inspection, the mother appeared unable to produce milk. This
suggests either that DDGAT is required for mammary gland
differentiation, or because the mammary gland resides in a fat pad,
DGAT deficiency-results in a decreased fat pad that is insufficient
to support mammary gland differentiation/milk production. The data
also indicate that mutations in the DGAT gene is involved in breast
cancer development. As such, this gene might be a target for
diagnosis and/or treatment.
[0184] 7. Aging Phenotype
[0185] The phenotype of the female mice, determined 5 weeks after
the second animal gave birth (to allow recovery; triglyceride
metabolism is altered in pregnancy and the post partum period) was
similar to that of the males in the second group, i.e. fur loss in
patches, especially in the intrascapular region. Again, very little
intrabdominal fat, very little subcutaneous fat and decreased
retroperitoneal fat pads were observed. Body weight was 12% lower
in the -/- female mice with no changes in serum cholesterol or
glucose levels, but with about a 50% decrease in triglycerides.
These data suggest that the animals become more affected with age.
This observation is likely due to a cumulative effect of decreased
fat absorption throughout adult life due to low DGAT activity in
the duodenum/jejunum; however, this may also be coupled to
decreased ability to store fat in adipose tissue due to adipocyte
DGAT-1 deficiency.
[0186] IV. Additional Studies
[0187] A. DGAT Overexpressing Transgenic Mice
[0188] DGAT-overexpressing transgenic mice were generated by
standard techniques using the mouse DGAT (now known as DGAT1) cDNA
and the aP2 promoter. Two transgenic lines in a C57BL/6 background
have been generated. Both overexpress DGAT1 in white adipose tissue
at levels approximately twice the level of the endogenous DGAT1
gene. Feeding these mice a high-fat diet results in mice that are
significantly heavier than wild type mice, indicating that
DGAT-overexpression in adipose tissue promotes obesity.
[0189] B. Generation of Knockout Cells
[0190] DGAT 1-deficient murine embryonic fibroblasts were generated
by culturing fibroblasts derived from Dgat-/- embryos. Several
lines have been generated and immortalized by sequential passaging.
Membranes from these Dgat-/- fibroblasts are deficient in DGAT
activity. These fibroblasts are useful for providing a
DGAT-deficient mammalian cell line for overexpressing human DGAT1
or DGAT2 to test pharmacologic inhibitors of the enzyme.
[0191] It is apparent from the above results and discussion that
polynucleotides encoding both animal and plant DGAT enzymes, as
well as novel polypeptides encoded thereby, are provided. The
subject invention is important for both research and therapeutic
applications. Using the DGAT probes of the subject invention, the
role of DGAT and its regulation in a number of physiological
processes can be studied in vivo. The subject invention also
provides for important new ways of treating diseases associated
with DGAT, such as hypertriglycemia and obesity, as well as in the
production of tryglycerides.
[0192] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0193] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
Sequence CWU 1
1
10 1 1411 DNA homo sapiens 1 ttatttttgg agaacctcat caagtatggc
atcctggtgg accccatcca ggtggtttct 60 ctgttcctga aggatcccta
tagctggccc gccccatgcc tggttattgc ggccaatgtt 120 tttgctgtgg
ctgcattcca ggttgagaag cgcctggcgg tgggtgccct gacggagcag 180
gcgggactgc tgctgcacgt ggccaacctg gccaccattc tgtgtttccc agcggctgtg
240 gtcttactgg ttgagtctat cactccagtg ggctccctgc tggcgctgat
ggcgcacacc 300 atcctcttcc tcaagctctt ctcctaccgc gacgtcaact
catggtgccg cagggccagg 360 gccaaggctg cctctgcagg gaagaaggcc
agcagtgttg ctgccccgca caccgtgagc 420 tacccggaca atctgaccta
ccgcgatctc tactacttcc tcttcgcccc caccttgtgc 480 tacgagctca
actttccccg ctctccccgc atccggaagc gctttctgct gcgacggatc 540
cttgagatgc tgttcttcac ccagctccag gtggggctga tccagcagtg gatggtcccc
600 accatccaga actccatgaa gcccttcaag gacatggact actcacgcat
catcgagcgc 660 ctcctgaagc tggcggtccc caatcacctc atctggctca
tcttcttcta ctggctcttc 720 cactcctgcc tgaatgccgt ggctgagctc
atgcagtttg gagaccggga gttctaccgg 780 gactggtgga actccgagtc
tgtcacctac ttctggcaga actggaacat ccctgtgcac 840 aagtggtgca
tcagacactt ctacaagccc atgcttcgac ggggcagcag caagtggatg 900
gccaggacag gggtgttcct ggcctcggcc ttcttccacg agtacctggt gagcgtccct
960 ctgcgaatgt tccgcctctg ggcgttcacg ggcatgatgg ctcagatccc
actggcctgg 1020 ttcgtgggcc gctttttcca gggcaactat ggcaacgcag
ctgtgtggct gtcgctcatc 1080 atcggacagc caatagccgt cctcatgtac
gtccacgact actacgtgct caactatgag 1140 gccccagcgg cagaggcctg
agctgcacct gaggggctgg cttctcactg ccacctcaca 1200 cccgctggca
gagcccacct ctcctcctag gcctcgagtt gctggggatg ggcctggctg 1260
cacagcatcc tcctctggtc ccagggaggc ctctctgccc ctatggggct ctgtcctgca
1320 cccctcaggg atggcgacag caggccagac acagtctgat gccagctggg
agtcttgctg 1380 accctgcccc gggtccgagg gtgtcaataa a 1411 2 261 DNA
homo sapiens 2 cagaggcctg agctgcacct gaggggctgg cttctcactg
ccacctcaca cccgctggca 60 gagcccacct ctcctcctag gcctcgagtt
gctggggatg ggcctggctg cacagcatcc 120 tcctctggtc ccagggaggc
ctctctgccc ctatggggct ctgtcctgca cccctcaggg 180 atggcgacag
caggccagac acagtctgat gccagctggg agtcttgctg accctgcccc 240
gggtccgagg gtgtcaataa a 261 3 1650 DNA mus musculus 3 ggatgaatgg
aaataagtag aattaggcat acttaggata gggctcaagc cgcggcccgt 60
gaagattggg ccgcgacgag gtgcgggccg aagccatggg cgaccgcgga ggcgcgggaa
120 gctctcggcg tcggaggacc ggctcgcggg tttccgtcca gggtggtagt
gggcccaagg 180 tagaagagga cgaggtgcga gacgcggctg tgagccccga
cttgggcgcc gggggtgacg 240 cgccggctcc ggctccggct ccagcccata
cccgggacaa agacgggcgg accagcgtgg 300 gcgacggcta ctgggatctg
aggtgccatc gtctgcaaga ttctttgttc agctcagaca 360 gtggtttcag
caattatcgt ggtatcctga attggtgtgt ggtgatgctg atcctgagta 420
atgcaaggtt atttttagag aaccttatca agtatggcat cctggtggat cctatccagg
480 tggtgtctct gtttttgaag gacccctaca gctggcctgc cccatgcgtg
attattgcat 540 ccaatatttt tgttgtggct gcatttcaga ttgagaagcg
cctggcagtg ggtgccctga 600 cagagcagat ggggctgctg ctacatgtgg
ttaacctggc cacaatcatt tgcttcccag 660 cagctgtggc cttactggtt
gagtctatca ctccagtggg ttccgtgttt gctctggcat 720 catactccat
catgttcctc aagctttatt cctaccggga tgtcaacctg tggtgccgcc 780
agcgaagggt caaggccaaa gctgtctcta cagggaagaa ggtcagtggg gctgctgccc
840 agcaagctgt gagctatcca gacaacctga cctaccgaga tctctattac
ttcatctttg 900 ctcctacttt gtgttatgaa ctcaactttc ctcggtcccc
cgcaatacga aagcgctttc 960 tgctacgacg agttcttgag atgctctttt
ttacccagct tcaagtgggg ctgatccaac 1020 agtggatggt ccctactatc
cacaactcca tgaagccctt caaggatatg gactattcac 1080 ggatcattga
gcgtctctta aagctggcgg tccccaacca tctgatctgg cttatcttct 1140
tctattggtt tttccactcc tgtctcaatg ctgtggcaga gcttctgcag tttggagacc
1200 gcgagttcta cagagattgg tggaatgctg agtctgtcac ctacttttgg
cagaactgga 1260 atatccccgt gcacaagtgg tgcatcagac acttctacaa
gcctatgctc agacatggca 1320 gcagcaaatg ggtggccagg acaggagtat
ttttgacctc agccttcttc catgagtacc 1380 tagtgagcgt tcccctgcgg
atgttccgcc tctgggcatt cacagccatg atggctcagg 1440 tcccactggc
ctggattgtg ggccgattct tccaagggaa ctatggcaat gcagctgtgt 1500
gggtgacact catcattggg caaccggtgg ctgtgctcat gtatgtccac gactactacg
1560 tgctcaacta cgatgcccca gtgggggtat gagctactgc caaaggccag
ccctccctaa 1620 cctgggcctg gagttctgga ggggttcctg 1650 4 629 DNA
arabidopsis thaliana misc_feature (0)...(0) Each n residue at
position 455, 464, 467, 475, 497, 500, 508, 514, 519, 536, 543,
544, 576, 583, 584 and 597 can be either a, c, g or t 4 tgcatgtata
cggaagggtt gggtggctcg tcaatttgca aaactggtca tattcaccgg 60
attcatggga tttataatag aacaatatat aaatcctatt gtcaggaact caaagcatcc
120 tttgaaaggc gatcttctat atgctattga aagagtgttg aagctttcag
ttccaaattt 180 atatgtgtgg ctctgcatgt tctactgctt cttccacctt
tggttaaaca tattggcaga 240 gcttctctgc ttcggggatc gtgaattcta
caaagattgg tggaatgcaa aaagtgtggg 300 agattactgg gagaatgtgg
aatatgcctg tccataaatg ggatgggtcc gacatatata 360 ccttccccgt
gcttgcgcac aaggattacc caaagacacc ccggccatta accattggct 420
ttcccaagcc ccctggaggc ctttccatgg gccanggacc cggngtnccc tggcnggccc
480 ttcaaagcaa agggggnttn cctggggnta aagntccang ggcccttggg
gcccanccaa 540 aannttcccc cgggaaaggg ttgcccaccg gggggngaaa
aanncccggg ggcaccncgg 600 aattttggga acccgggggg ggccttttt 629 5 386
PRT homo sapiens 5 Leu Phe Leu Glu Asn Leu Ile Lys Tyr Gly Ile Leu
Val Asp Pro Ile 1 5 10 15 Gln Val Val Ser Leu Phe Leu Lys Asp Pro
Tyr Ser Trp Pro Ala Pro 20 25 30 Cys Leu Val Ile Ala Ala Asn Val
Phe Ala Val Ala Ala Phe Gln Val 35 40 45 Glu Lys Arg Leu Ala Val
Gly Ala Leu Thr Glu Gln Ala Gly Leu Leu 50 55 60 Leu His Val Ala
Asn Leu Ala Thr Ile Leu Cys Phe Pro Ala Ala Val 65 70 75 80 Val Leu
Leu Val Glu Ser Ile Thr Pro Val Gly Ser Leu Leu Ala Leu 85 90 95
Met Ala His Thr Ile Leu Phe Leu Lys Leu Phe Ser Tyr Arg Asp Val 100
105 110 Asn Ser Trp Cys Arg Arg Ala Arg Ala Lys Ala Ala Ser Ala Gly
Lys 115 120 125 Lys Ala Ser Ser Val Ala Ala Pro His Thr Val Ser Tyr
Pro Asp Asn 130 135 140 Leu Thr Tyr Arg Asp Leu Tyr Tyr Phe Leu Phe
Ala Pro Thr Leu Cys 145 150 155 160 Tyr Glu Leu Asn Phe Pro Arg Ser
Pro Arg Ile Arg Lys Arg Phe Leu 165 170 175 Leu Arg Arg Ile Leu Glu
Met Leu Phe Phe Thr Gln Leu Gln Val Gly 180 185 190 Leu Ile Gln Gln
Trp Met Val Pro Thr Ile Gln Asn Ser Met Lys Pro 195 200 205 Phe Lys
Asp Met Asp Tyr Ser Arg Ile Ile Glu Arg Leu Leu Lys Leu 210 215 220
Ala Val Pro Asn His Leu Ile Trp Leu Ile Phe Phe Tyr Trp Leu Phe 225
230 235 240 His Ser Cys Leu Asn Ala Val Ala Glu Leu Met Gln Phe Gly
Asp Arg 245 250 255 Glu Phe Tyr Arg Asp Trp Trp Asn Ser Glu Ser Val
Thr Tyr Phe Trp 260 265 270 Gln Asn Trp Asn Ile Pro Val His Lys Trp
Cys Ile Arg His Phe Tyr 275 280 285 Lys Pro Met Leu Arg Arg Gly Ser
Ser Lys Trp Met Ala Arg Thr Gly 290 295 300 Val Phe Leu Ala Ser Ala
Phe Phe His Glu Tyr Leu Val Ser Val Pro 305 310 315 320 Leu Arg Met
Phe Arg Leu Trp Ala Phe Thr Gly Met Met Ala Gln Ile 325 330 335 Pro
Leu Ala Trp Phe Val Gly Arg Phe Phe Gln Gly Asn Tyr Gly Asn 340 345
350 Ala Ala Val Trp Leu Ser Leu Ile Ile Gly Gln Pro Ile Ala Val Leu
355 360 365 Met Tyr Val His Asp Tyr Tyr Val Leu Asn Tyr Glu Ala Pro
Ala Ala 370 375 380 Glu Ala 385 6 488 PRT homo sapiens 6 Met Gly
Asp Arg Gly Ser Ser Arg Arg Arg Arg Thr Gly Ser Arg Pro 1 5 10 15
Ser Ser His Gly Gly Gly Gly Pro Ala Ala Ala Glu Glu Glu Val Arg 20
25 30 Asp Ala Ala Ala Gly Pro Asp Val Gly Ala Ala Gly Asp Ala Pro
Ala 35 40 45 Pro Ala Pro Asn Lys Asp Gly Asp Ala Gly Val Gly Ser
Gly His Trp 50 55 60 Glu Leu Arg Cys His Arg Leu Gln Asp Ser Leu
Phe Ser Ser Asp Ser 65 70 75 80 Gly Phe Ser Asn Tyr Arg Gly Ile Leu
Asn Trp Cys Val Val Met Leu 85 90 95 Ile Leu Ser Asn Ala Arg Leu
Phe Leu Glu Asn Leu Ile Lys Tyr Gly 100 105 110 Ile Leu Val Asp Pro
Ile Gln Val Val Ser Leu Phe Leu Lys Asp Pro 115 120 125 His Ser Trp
Pro Ala Pro Cys Leu Val Ile Ala Ala Asn Val Phe Ala 130 135 140 Val
Ala Ala Phe Gln Val Glu Lys Arg Leu Ala Val Gly Ala Leu Thr 145 150
155 160 Glu Gln Ala Gly Leu Leu Leu His Val Ala Asn Leu Ala Thr Ile
Leu 165 170 175 Cys Phe Pro Ala Ala Val Val Leu Leu Val Glu Ser Ile
Thr Pro Val 180 185 190 Gly Ser Leu Leu Ala Leu Met Ala His Thr Ile
Leu Phe Leu Lys Leu 195 200 205 Phe Ser Tyr Arg Asp Val Asn Ser Trp
Cys Arg Arg Ala Arg Ala Lys 210 215 220 Ala Ala Ser Ala Gly Lys Lys
Ala Ser Ser Ala Ala Ala Pro His Thr 225 230 235 240 Val Ser Tyr Pro
Asp Asn Leu Thr Tyr Arg Asp Leu Tyr Tyr Phe Leu 245 250 255 Phe Ala
Pro Thr Leu Cys Tyr Glu Leu Asn Phe Pro Arg Ser Pro Arg 260 265 270
Ile Arg Lys Arg Phe Leu Leu Arg Arg Ile Leu Glu Met Leu Phe Phe 275
280 285 Thr Gln Leu Gln Val Gly Leu Ile Gln Gln Trp Met Val Pro Thr
Ile 290 295 300 Gln Asn Ser Met Lys Pro Phe Lys Asp Met Asp Tyr Ser
Arg Ile Ile 305 310 315 320 Glu Arg Leu Leu Lys Leu Ala Val Pro Asn
His Leu Ile Trp Leu Ile 325 330 335 Phe Phe Tyr Trp Leu Phe His Ser
Cys Leu Asn Ala Val Ala Glu Leu 340 345 350 Met Gln Phe Gly Asp Arg
Glu Phe Tyr Arg Asp Trp Trp Asn Ser Glu 355 360 365 Ser Val Thr Tyr
Phe Trp Gln Asn Trp Asn Ile Pro Val His Lys Trp 370 375 380 Cys Ile
Arg His Phe Tyr Lys Pro Met Leu Arg Arg Gly Ser Ser Lys 385 390 395
400 Trp Met Ala Arg Thr Gly Val Phe Leu Ala Ser Ala Phe Phe His Glu
405 410 415 Tyr Leu Val Ser Val Pro Leu Arg Met Phe Arg Leu Trp Ala
Phe Thr 420 425 430 Gly Met Met Ala Gln Ile Pro Leu Ala Trp Phe Val
Gly Arg Phe Phe 435 440 445 Gln Gly Asn Tyr Gly Asn Ala Ala Val Trp
Leu Ser Leu Ile Ile Gly 450 455 460 Gln Pro Ile Ala Val Leu Met Tyr
Val His Asp Tyr Tyr Val Leu Asn 465 470 475 480 Tyr Glu Ala Pro Ala
Ala Glu Ala 485 7 498 PRT mus musculus 7 Met Gly Asp Arg Gly Gly
Ala Gly Ser Ser Arg Arg Arg Arg Thr Gly 1 5 10 15 Ser Arg Val Ser
Val Gln Gly Gly Ser Gly Pro Lys Val Glu Glu Asp 20 25 30 Glu Val
Arg Asp Ala Ala Val Ser Pro Asp Leu Gly Ala Gly Gly Asp 35 40 45
Ala Pro Ala Pro Ala Pro Ala Pro Ala His Thr Arg Asp Lys Asp Gly 50
55 60 Arg Thr Ser Val Gly Asp Gly Tyr Trp Asp Leu Arg Cys His Arg
Leu 65 70 75 80 Gln Asp Ser Leu Phe Ser Ser Asp Ser Gly Phe Ser Asn
Tyr Arg Gly 85 90 95 Ile Leu Asn Trp Cys Val Val Met Leu Ile Leu
Ser Asn Ala Arg Leu 100 105 110 Phe Leu Glu Asn Leu Ile Lys Tyr Gly
Ile Leu Val Asp Pro Ile Gln 115 120 125 Val Val Ser Leu Phe Leu Lys
Asp Pro Tyr Ser Trp Pro Ala Pro Cys 130 135 140 Val Ile Ile Ala Ser
Asn Ile Phe Val Val Ala Ala Phe Gln Ile Glu 145 150 155 160 Lys Arg
Leu Ala Val Gly Ala Leu Thr Glu Gln Met Gly Leu Leu Leu 165 170 175
His Val Val Asn Leu Ala Thr Ile Ile Cys Phe Pro Ala Ala Val Ala 180
185 190 Leu Leu Val Glu Ser Ile Thr Pro Val Gly Ser Val Phe Ala Leu
Ala 195 200 205 Ser Tyr Ser Ile Met Phe Leu Lys Leu Tyr Ser Tyr Arg
Asp Val Asn 210 215 220 Leu Trp Cys Arg Gln Arg Arg Val Lys Ala Lys
Ala Val Ser Thr Gly 225 230 235 240 Lys Lys Val Ser Gly Ala Ala Ala
Gln Gln Ala Val Ser Tyr Pro Asp 245 250 255 Asn Leu Thr Tyr Arg Asp
Leu Tyr Tyr Phe Ile Phe Ala Pro Thr Leu 260 265 270 Cys Tyr Glu Leu
Asn Phe Pro Arg Ser Pro Arg Ile Arg Lys Arg Phe 275 280 285 Leu Leu
Arg Arg Val Leu Glu Met Leu Phe Phe Thr Gln Leu Gln Val 290 295 300
Gly Leu Ile Gln Gln Trp Met Val Pro Thr Ile His Asn Ser Met Lys 305
310 315 320 Pro Phe Lys Asp Met Asp Tyr Ser Arg Ile Ile Glu Arg Leu
Leu Lys 325 330 335 Leu Ala Val Pro Asn His Leu Ile Trp Leu Ile Phe
Phe Tyr Trp Phe 340 345 350 Phe His Ser Cys Leu Asn Ala Val Ala Glu
Leu Leu Gln Phe Gly Asp 355 360 365 Arg Glu Phe Tyr Arg Asp Trp Trp
Asn Ala Glu Ser Val Thr Tyr Phe 370 375 380 Trp Gln Asn Trp Asn Ile
Pro Val His Lys Trp Cys Ile Arg His Phe 385 390 395 400 Tyr Lys Pro
Met Leu Arg His Gly Ser Ser Lys Trp Val Ala Arg Thr 405 410 415 Gly
Val Phe Leu Thr Ser Ala Phe Phe His Glu Tyr Leu Val Ser Val 420 425
430 Pro Leu Arg Met Phe Arg Leu Trp Ala Phe Thr Ala Met Met Ala Gln
435 440 445 Val Pro Leu Ala Trp Ile Val Gly Arg Phe Phe Gln Gly Asn
Tyr Gly 450 455 460 Asn Ala Ala Val Trp Val Thr Leu Ile Ile Gly Gln
Pro Val Ala Val 465 470 475 480 Leu Met Tyr Val His Asp Tyr Tyr Val
Leu Asn Tyr Asp Ala Pro Val 485 490 495 Gly Val 8 10 PRT Artificial
Sequence synthetic peptide-FLAG epitope 8 Met Gly Asp Tyr Lys Asp
Asp Asp Asp Gly 1 5 10 9 1650 DNA mus musculus 9 ggatgaatgg
aaataagtag aattaggcat acttaggata gggctcaagc cgcggcccgt 60
gaagattggg ccgcgacgag gtgcgggccg aagccatggg cgaccgcgga ggcgcgggaa
120 gctctcggcg tcggaggacc ggctcgcggg tttccgtcca gggtggtagt
gggcccaagg 180 tagaagagga cgaggtgcga gacgcggctg tgagccccga
cttgggcgcc gggggtgacg 240 cgccggctcc ggctccggct ccagcccata
cccgggacaa agacgggcgg accagcgtgg 300 gcgacggcta ctgggatctg
aggtgccatc gtctgcaaga ttctttgttc agctcagaca 360 gtggtttcag
caattatcgt ggtatcctga attggtgtgt ggtgatgctg atcctgagta 420
atgcaaggtt atttttagag aaccttatca agtatggcat cctggtggat cctatccagg
480 tggtgtctct gtttttgaag gacccctaca gctggcctgc cccatgcgtg
attattgcat 540 ccaatatttt tgttgtggct gcatttcaga ttgagaagcg
cctggcagtg ggtgccctga 600 cagagcagat ggggctgctg ctacatgtgg
ttaacctggc cacaatcatt tgcttcccag 660 cagctgtggc cttactggtt
gagtctatca ctccagtggg ttccgtgttt gctctggcat 720 catactccat
catgttcctc aagctttatt cctaccggga tgtcaacctg tggtgccgcc 780
agcgaagggt caaggccaaa gctgtctcta cagggaagaa ggtcagtggg gctgctgccc
840 agcaagctgt gagctatcca gacaacctga cctaccgaga tctctattac
ttcatctttg 900 ctcctacttt gtgttatgaa ctcaactttc ctcggtcccc
cgcaatacga aagcgctttc 960 tgctacgacg agttcttgag atgctctttt
ttacccagct tcaagtgggg ctgatccaac 1020 agtggatggt ccctactatc
cacaactcca tgaagccctt caaggatatg gactattcac 1080 ggatcattga
gcgtctctta aagctggcgg tccccaacca tctgatctgg cttatcttct 1140
tctattggtt tttccactcc tgtctcaatg ctgtggcaga gcttctgcag tttggagacc
1200 gcgagttcta cagagattgg tggaatgctg agtctgtcac ctacttttgg
cagaactgga 1260 atatccccgt gcacaagtgg tgcatcagac acttctacaa
gcctatgctc agacatggca 1320 gcagcaaatg ggtggccagg acaggagtat
ttttgacctc agccttcttc catgagtacc 1380 tagtgagcgt tcccctgcgg
atgttccgcc tctgggcatt cacagccatg atggctcagg 1440 tcccactggc
ctggattgtg ggccgattct tccaagggaa ctatggcaat gcagctgtgt 1500
gggtgacact catcattggg caaccggtgg ctgtgctcat gtatgtccac gactactacg
1560 tgctcaacta cgatgcccca gtgggggtat gagctactgc caaaggccag
ccctccctaa 1620 cctgggcctg gagttctgga ggggttcctg 1650 10 498 PRT
mus musculus 10 Met Gly Asp Arg Gly Gly Ala Gly Ser Ser Arg Arg Arg
Arg Thr Gly 1 5 10 15 Ser Arg Val Ser Val Gln Gly Gly Ser Gly Pro
Lys Val Glu Glu Asp 20 25 30 Glu Val Arg Asp Ala Ala Val Ser Pro
Asp Leu Gly Ala Gly Gly Asp 35 40 45 Ala Pro Ala Pro Ala Pro Ala
Pro Ala His Thr Arg Asp Lys Asp Gly 50 55 60 Arg Thr Ser Val Gly
Asp Gly Tyr Trp Asp Leu Arg Cys His Arg Leu
65 70 75 80 Gln Asp Ser Leu Phe Ser Ser Asp Ser Gly Phe Ser Asn Tyr
Arg Gly 85 90 95 Ile Leu Asn Trp Cys Val Val Met Leu Ile Leu Ser
Asn Ala Arg Leu 100 105 110 Phe Leu Glu Asn Leu Ile Lys Tyr Gly Ile
Leu Val Asp Pro Ile Gln 115 120 125 Val Val Ser Leu Phe Leu Lys Asp
Pro Tyr Ser Trp Pro Ala Pro Cys 130 135 140 Val Ile Ile Ala Ser Asn
Ile Phe Val Val Ala Ala Phe Gln Ile Glu 145 150 155 160 Lys Arg Leu
Ala Val Gly Ala Leu Thr Glu Gln Met Gly Leu Leu Leu 165 170 175 His
Val Val Asn Leu Ala Thr Ile Ile Cys Phe Pro Ala Ala Val Ala 180 185
190 Leu Leu Val Glu Ser Ile Thr Pro Val Gly Ser Val Phe Ala Leu Ala
195 200 205 Ser Tyr Ser Ile Met Phe Leu Lys Leu Tyr Ser Tyr Arg Asp
Val Asn 210 215 220 Leu Trp Cys Arg Gln Arg Arg Val Lys Ala Lys Ala
Val Ser Thr Gly 225 230 235 240 Lys Lys Val Ser Gly Ala Ala Ala Gln
Gln Ala Val Ser Tyr Pro Asp 245 250 255 Asn Leu Thr Tyr Arg Asp Leu
Tyr Tyr Phe Ile Phe Ala Pro Thr Leu 260 265 270 Cys Tyr Glu Leu Asn
Phe Pro Arg Ser Pro Arg Ile Arg Lys Arg Phe 275 280 285 Leu Leu Arg
Arg Val Leu Glu Met Leu Phe Phe Thr Gln Leu Gln Val 290 295 300 Gly
Leu Ile Gln Gln Trp Met Val Pro Thr Ile His Asn Ser Met Lys 305 310
315 320 Pro Phe Lys Asp Met Asp Tyr Ser Arg Ile Ile Glu Arg Leu Leu
Lys 325 330 335 Leu Ala Val Pro Asn His Leu Ile Trp Leu Ile Phe Phe
Tyr Trp Phe 340 345 350 Phe His Ser Cys Leu Asn Ala Val Ala Glu Leu
Leu Gln Phe Gly Asp 355 360 365 Arg Glu Phe Tyr Arg Asp Trp Trp Asn
Ala Glu Ser Val Thr Tyr Phe 370 375 380 Trp Gln Asn Trp Asn Ile Pro
Val His Lys Trp Cys Ile Arg His Phe 385 390 395 400 Tyr Lys Pro Met
Leu Arg His Gly Ser Ser Lys Trp Val Ala Arg Thr 405 410 415 Gly Val
Phe Leu Thr Ser Ala Phe Phe His Glu Tyr Leu Val Ser Val 420 425 430
Pro Leu Arg Met Phe Arg Leu Trp Ala Phe Thr Ala Met Met Ala Gln 435
440 445 Val Pro Leu Ala Trp Ile Val Gly Arg Phe Phe Gln Gly Asn Tyr
Gly 450 455 460 Asn Ala Ala Val Trp Val Thr Leu Ile Ile Gly Gln Pro
Val Ala Val 465 470 475 480 Leu Met Tyr Val His Asp Tyr Tyr Val Leu
Asn Tyr Asp Ala Pro Val 485 490 495 Gly Val
* * * * *
References